Biomedical subjects

C Englert

Publications and source records attributed to C Englert.

36 records · Page 2Linked to original sources

PubMed1996

Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein.

A tetracycline-regulated system was used to generate cell lines allowing tightly controlled expression of a hepatitis C virus (HCV) cDNA comprising the 5' noncoding, the core, and part of the E1 regions. Production of 21-kDa processed nucleocapsid protein could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Induction ratios of over 1,000-fold were found using an HCV core-luciferase fusion construct. Core protein had an intracellular half-life of 9 hr and corresponded to the product of 173 amino-terminal amino acids of the HCV open reading frame. Sequential immunofluorescence microscopy revealed the presence of core antigen first in a predominantly perinuclear fine-reticular staining pattern and subsequently also in cytoplasmic granules and vesicles. By immunoelectron microscopy core protein was found on the endoplasmic reticulum membrane and on the surface of cytoplasmic lipid droplets. Growth rate analyses and colony formation efficiency assays showed no major cytotoxic effect of HCV core protein expression per se. HCV gene expression could be inhibited by an antisense oligonucleotide targeting a region immediately downstream of the translation initiation codon. These cell lines represent important tools to investigate structural and functional properties of HCV core protein and may be useful to evaluate gene therapeutic strategies against HCV in a cellular system.

Cell Division↗

PubMed1996

Germ-line BRCA1 mutations in Jewish and non-Jewish women with early-onset breast cancer.

BACKGROUND: Mutations in a germ-line allele of the BRCA1 gene contribute to the familial breast cancer syndrome. However, the prevalence of these mutations is unknown in women with breast cancer who do not have the features of this familial syndrome. We sought BRCA1 mutations in women who were given a diagnosis of breast cancer at an early age, because early onset is characteristic of a genetic predisposition to cancer. METHODS: Clinical information and peripheral-blood mononuclear cells were obtained from 418 women from the Boston metropolitan area in whom breast cancer was diagnosed at or before the age of 40. A comprehensive BRCA1 mutational analysis, involving automated nucleotide sequencing and a protein-truncation assay, was undertaken in 30 of these women, who had breast cancer before the age of 30. In addition, the BRCA1 mutation 185delAG, which is prevalent in the Ashkenazi Jewish population, was sought with an allele-specific polymerase-chain-reaction assay in 39 Jewish women among the 418 women who had breast cancer at or before the age of 40. RESULTS: Among 30 women with breast cancer before the age of 30, 4 (13 percent) had definite, chain-terminating mutations and 1 had a missense mutation. Two of the four Jewish women in this cohort had the 185delAG mutation. Among the 39 Jewish women with breast cancer at or before the age of 40, 8 (21 percent) carried the 185delAG mutation (95 percent confidence interval, 9 to 36 percent). CONCLUSIONS: Germ-line BRCA1 mutations can be present in young women with breast cancer who do not belong to families with multiple affected members. The specific BRCA1 mutation known as 185delAG is strongly associated with the onset of breast cancer in Jewish women before the age of 40.

Adult↗

PubMed1996

Functional properties of WT1.

WT1 encodes a zinc finger transcription factor that is inactivated in a subset of Wilms' tumors. We have recently shown that introduction of wild-type WT1 into a Wilms' tumor-derived cell line, RM1, results in growth suppression, consistent with its function as a tumor suppressor gene. WT1-mediated growth suppression was also observed in other cells derived from embryonal tumors, including two osteosarcoma cell lines, U2OS and Saos-2, notable for the respective presence or absence of wild-type p53. To further characterize the functional properties of WT1, multiple U2OS and Saos-2 cell lines were established, expressing either wild-type WT1 splicing variants or naturally occurring mutants under control of a tightly regulated tetracycline repressable promoter. Induction of WT1 in these cells resulted in programmed cell death. This effect was preferentially mediated by WT1 isoform B (encoding alternative splice I, lacking alternative splice II "KTS"), and it was independent of p53, occurring in both U2OS and Saos-2 cells. WT1-mediated apoptosis was associated with transcriptional repression of the epidermal growth factor receptor (EGFR) and reduced synthesis of endogenous EGFR protein synthesis. Constitutive expression of EGFR abrogated WT1-mediated cell death. We conclude that wild-type WT1 can induce apoptosis in embryonal cancer cells, presumably through the withdrawal of required growth factor survival signals, and that EGFR is a physiological target gene for WT1.

Alternative Splicing↗

PubMed1996

A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Amino Acid Sequence↗

PubMed1995

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

Binding Sites↗

PubMed1995

WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

Alternative Splicing↗

PubMed1995

The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis.

The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.

Animals↗

PubMed1995

Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.

Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.

Amino Acid Sequence↗

PubMed1993

WT1-mediated growth suppression of Wilms tumor cells expressing a WT1 splicing variant.

A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.

Alternative Splicing↗

PubMed1993

Analysis of gas vesicle gene expression in Haloferax mediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins.

Gas vesicle synthesis in Haloferax mediterranei involves several gene products encoded by a 9.4-kilobase pair DNA region (mc-vac region) that contains 13 genes in addition to gvpA encoding the major structural gas vesicle protein. The expression of part of this region, encompassing the genes gvpA, gvpC, gvpN, and gvpO was investigated. These genes are transcribed from a common promoter located upstream of gvpA. Transcripts of 0.34 (gvpA only), 1.8 (gvpA/C), 2.4 (gvpA/C/N) and 3 kilobases (gvpA/C/N/O) were observed, with the gvpA transcript being the predominant mRNA species. The majority of the mRNA formed terminates 64 base pairs downstream of gvpA at the cytosine of the sequence 5' TTTTTC 3'. The synthesis of the GvpA and GvpC proteins was investigated by Western analyses. An antiserum raised against isolated gas vesicles of Hf. mediterranei detects, in addition to gas vesicle fragments, the GvpA protein of the M(r) of approximately 8,000 in lysates derived from different halobacteria or from Escherichia coli expressing gvpA. In samples containing isolated gas vesicles, mainly partially disaggregated gas vesicle fragments hybridize, but a minor amount of monomeric GvpA is also seen. For the detection of the GvpC protein, two versions of the gvpC gene (full length and gvp delta C lacking the 3' part encoding the acidic C terminus) were expressed in E. coli, and the resulting proteins were purified. The two antisera raised against these GvpC versions indicate the expression of gvpC in different halobacteria. By Western analysis, GvpC is also detectable in samples containing isolated gas vesicles demonstrating that GvpC is a second, but minor, gas vesicle structural protein.

Amino Acid Sequence↗

PubMed1992

Three different but related gene clusters encoding gas vesicles in halophilic archaea.

We present an analysis of the chromosomal region comprising the gene cluster involved in gas vesicle (Vac) synthesis in Haloferax mediterranei (mc-vac-region) and Halobacterium salinarium (c-vac-region) and compare both of them to the plasmid located p-vac-region of H. salinarium. The p-vac-region of 9000 base-pairs (9 kb) is more related to mc-vac (9.4 kb) of Hf. mediterranei than it is to the c-vac-region (8.3 kb) present in the same cell. The Vac- species Hf. volcanii becomes Vac+ following transformation with a fragment containing the entire mc-vac-region. Also the p-vac-region transforms Hf. volcanii to a Vac+ phenotype, indicating that this gene cluster is sufficient for gas vesicle synthesis and does not depend on products of the c-vac-region. Each of these vac-regions contains, in addition to gvpA encoding the major gas vesicle protein, 13 open reading frames named gvpC through gvpO. Ten of these, gvpD through gvpM, are located upstream from gvpA in opposite orientation, while gvpC, gvpN and gvpO are found 3' to gvpA. The absolute requirement of gvpO for gas vesicle synthesis was demonstrated by transformation experiments. Northern analyses with RNA samples isolated during the growth cycle of Hf. mediterranei or of H. salinarium PHH4 revealed that the mc-gvpD or c-gvpD mRNAs occur similar to the respective gvpA mRNA in stationary growth phase, while gvpF-gvpM are transcribed mainly during logarithmic growth. S1-nuclease mapping was performed to determine the transcriptional start site of the gvpD mRNA. The distance between the two divergent start sites of gvpA and gvpD mRNA is 109 base-pairs in mc-vac and p-vac, while in the case of c-vac this distance is 22 base-pairs larger. The conservation of the various gvp products, characteristic features and their possible functions in gas vesicle synthesis are discussed.

Archaeal Proteins↗

PubMed1992

Function and biosynthesis of gas vesicles in halophilic Archaea.

The proteinaceous gas vesicles produced by various microorganisms including halophilic Archaea are hollow, gas-filled structures with a hydrophobic inner and a hydrophilic outer surface. The structural components of gas vesicles and their biosynthesis are still under investigation; an 8-kDa polypeptide appears to be the major constituent of the gas-vesicle envelope. Genetic analysis of the halobacterial gas-vesicle synthesis revealed an unexpected complexity: about 14 genes organized in three transcription units are involved in gas-vesicle structure, assembly, and gene regulation. Here we describe the comparison of three different genomic regions encoding gas vesicles in Halobacterium salinarium (p-vac and c-vac regions) and Haloferax mediterranei (mc-vac region) and speculate on the function of the gene products involved in gas-vesicle synthesis.

Amino Acid Sequence↗

PubMed1992

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product.

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.

Bacterial Proteins↗

PubMed1991

A DNA region of 9 kbp contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria.

We determined the minimal size of the genomic region necessary for gas vesicle synthesis in halophilic archaebacteria by transformation experiments, comparative DNA sequence analysis and investigation of gas vesicle (Vac) mutants. The comparison of the three genomic regions encoding gas vesicles in Halobacterium halobium (p-vac- and c-vac-region) and Haloferax mediterranei (mc-vac-region) indicates high DNA sequence similarity throughout a contiguous sequence of 9 kbp. In each case, this area encompassed at least 13 open reading frames (ORFs). Ten of these ORFs (gvpD to gvpM) were located 5' to the vac gene encoding the major gas vesicle protein, but were transcribed from the opposite strand. At least two ORFs (gvpC, and gvpN) were located 3' to each vac gene and transcribed from the same strand as the respective vac gene. In the p-vac-region present on plasmid pHH1 these ORFs were transcribed as at least three units, one transcript encompassing gvpD-gvpE, the second encompassing ORFs gvpF to gvpM, and the third unit comprising the ORFs located 3' to the p-vac gene. In H. halobium Vac mutants copies of the insertion elements ISH2, ISH23, ISH26 or ISH27 were found to be integrated throughout the p-vac-region. The de novo synthesis of gas vesicles was tested by transformation of the Vac-negative species, Haloferax volcanii, with various subfragments of the mc-vac- or p-vac-region cloned into vector plasmids. In contrast to a fragment containing the entire 9 kbp region, none of the subfragments tested was sufficient to promote gas vesicle synthesis. However, gas vesicle synthesis could be restored in each Vac mutant containing an ISH element when the entire transcription unit encompassing the mutated gene on pHH1 was present in the wild-type form on the vector construct.

Amino Acid Sequence↗

PubMed1990

Expression of the major gas vesicle protein gene in the halophilic archaebacterium Haloferax mediterranei is modulated by salt.

In the moderately to extremely halophilic archaebacterium Haloferax mediterranei gas vacuoles are not observed before the stationary phase of growth, and only when the cells are grown in media containing more than 17% total salt. Under the electron microscope, isolated gas vesicles appear as cylindrical structures with conical ends that reach a maximal length of 1.5 microns; this morphology is different from the spindle-shaped gas vesicles found in the Halobacterium halobium wild type which expresses the plasmid-borne p-vac gene, but resembles that of gas vesicles isolated from H. halobium strains expressing the chromosomal c-vac gene. Both the p-vac and the c-vac genes encode very similar structural proteins accounting for the major part of the "membrane" of the respective gas vesicles. The homologous mc-vac gene was isolated from Hf. mediterranei using the p-vac gene as probe. The mc-vac coding region indicates numerous nucleotide differences compared to the p-vac anc c-vac genes; the encoded protein is, however, almost identical to the c-vac gene product. The start point of the 310 nucleotide mc-vac transcript determined by primer extension analysis and S1 mapping was located 20 bp upstream of the ATG start codon, which is at the same relative position as found for the other two vac mRNAs. During the growth cycle, mc-vac mRNA was detectable in Hf. mediterranei cells grown in 15% as well as 25% total salt, with a maximal level in the early stationary phase of growth. The relative abundance of mc-vac mRNA in cells grown at 25% salt was sevenfold higher than in cells grown in 15% total salt.

Amino Acid Sequence↗

PubMed1988

Two genes encoding gas vacuole proteins in Halobacterium halobium.

The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G + C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.

Amino Acid Sequence↗

PubMedDate not supplied

The Wilms tumor suppressor gene wt1 is required for development of the spleen.

The Wilms tumor suppressor gene WT1 (wt1 in mouse) is unique among tumor suppressors because, in addition to its involvement in cancer [1] [2] and various other diseases [3] [4] [5] [6], it has an essential role in the development of certain organs. This is revealed by the phenotype of mice with inactivated wt1 alleles [7]. These animals exhibit a complete failure of kidney and gonad development as well as abnormalities of the heart and mesothelial structures. On a C57BL/6 genetic background, wt1(-/-) animals die between day 13.5 (E13.5) and 15.5 (E15.5) of embryonic development [7]. We report here that crossing of the wt1 mutation onto different mouse backgrounds delayed embryonic lethality until birth. In wt1(-/-) mice on these different genetic backgrounds, we observed a dramatic failure of spleen development, in addition to the well characterized phenotypic abnormalities. The spleen anlage formed at around E12 to E13 and involuted by the E15 stage, before the invasion of hematopoietic cells. The absence of proper spleen development in these wt1(-/-) embryos correlated with enhanced apoptosis in the primordial spleen cells. The expression of hox11, a gene that also controls development of the spleen [8] [9], was not altered by the inactivation of wt1. In situ hybridization revealed that the two genes are regulated independently. These findings demonstrate that the penetrance of the wt1(-/-) phenotype depends on the existence of one or more modifier gene(s) and that wt1 plays a pivotal role in the development of the spleen, thereby extending its role in organogenesis.

Animals↗

PubMedDate not supplied

Independent regulation of two separate gene activities in a continuous human cell line.

Tetracycline- and ecdysone-responsive systems have recently been developed for regulated gene expression in eukaryotic cells. Using nucleolar-targeted luciferase and beta-galactosidase as reporter proteins we demonstrate that both systems can function simultaneously and independently in a continuous human cell line. Both gene activities could be regulated over a broad range and at the single cell level by the concentrations of tetracycline and muristerone A, respectively, in the culture medium. The strategy described here will allow to investigate the function and interaction of two separate gene activities in a well-defined and reproducible cellular context.

Cell Line↗