Intracellular location and nuclear targeting of the Spi-1, Spi-2 and Spi-3 gene-derived serine protease inhibitors in non-secretory cells.

Proteases and their inhibitors are indispensable for the regulated activation and/or degradation of structural and functional proteins involved in basic cellular processes, e.g. in cell cycle control, cell growth, differentiation and apoptosis. In this context the serine protease inhibitors derived from the murine Spi-1, Spi-2 and Spi-3 genes, and their human homologs, deserve reconsideration. Microsequencing data indicate that a fraction of the three serpins has the capability to constitute a well characterized proteinase K, high salt and SDS-stable complex which coisolates with DNA under salting out conditions from various cell and tissue types. This tight association with DNA isolated under conditions designed to deproteinize DNA efficiently points to an in situ preformed chromatin complex. Accordingly, in addition to their well known functions as 'serum protease inhibitors' the Spi-1 and Spi-2 gene-derived proteins appear to have intracellular functions as well. The involvement of the three serpins in chromatin complexes requires their nuclear translocation. Application of (enhanced) green fluorescent protein technology and optical section microscopy reveals that truncation of the N-terminal signal sequences of the Spi-1 and Spi-2 gene-encoded proteins is a prerequisite for their nuclear translocation while non-truncated fusion proteins are enriched at the nuclear indentation which is the site of the Golgi apparatus and the centrosome. The identification of new species of intracellular serpins is of potential interest with respect to accumulating evidence for serine protease inhibitor-dependent inhibition or prevention of apoptosis.

Promoter of the gene encoding the 16 kDa DNA-binding and apoptosis-inducing C1D protein.

The 5' region of the gene encoding the human 16 kDa DNA-binding and apoptosis-inducing C1D protein was analysed for promoter activity. Sections of this region were cloned into a promoterless vector containing the enhanced green fluorescent protein (EGFP) as reporter gene. Expressed EGFP was estimated in transfected cells by quantitative fluorescence microscopy. The sequence between mRNA positions ATG -868 and ATG -12 results in relatively highest EGFP expression in transiently transfected human and murine cells. The upstream segment immediately adjacent to the 5' end of the most active fragment was identified as an inverted LINE-1 repeat element. Transient transfection experiments point to the presence of cis-acting repressing sequences on this LINE-1 element which reduce the transcriptional activity of the basal C1D promoter in human and murine cells by more than 95%. This result supports previous evidence suggesting that LINE-1 sequences may function as regulatory elements to control the expression of nearby genes.

One single mRNA encodes the centrosomal protein CCD41 and the endothelial cell protein C receptor (EPCR).

The cDNA encoding the centrosomal protein CCD41 is identical with the cDNA for the endothelial cell protein C receptor. This finding is not due to an artefact, e.g. caused by selection of false positive clones. The segment of the CCD41 cDNA encoding the protein originally termed CCD41 and deletion mutants of it were fused with the nucleotide sequence encoding the enhanced green fluorescent protein (EGFP). Transfection and expression of the full length construct produces a fusion protein mainly located in cell membranes reflecting the receptor-type protein. Deletion mutants, e.g. those where the signal sequence is deleted, result in fusion proteins which are exclusively incorporated into a small perinuclear structure which is the site of the centrosome. This result suggests that post-translational modification, namely deletion of the signal sequence, is decisive for the centrosomal location of the resulting centrosomal protein while the unprocessed protein is incorporated into cell membranes.

Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D.

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.

cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.

Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.

A multifunctional protein: involvement of the alpha-1 serum protease inhibitor in SDS and high salt-stable DNA-protein complexes.

Occasionally new and intriguing roles arise for proteins with well established functions. The alpha-1 serum protease inhibitor (alpha-1 PI) represents another example. Sequence identities exist in the alpha-1 PI and in a nuclear 52-kDa glycoprotein which is involved in resistant DNA-polypeptide complexes. The results of Western blots support the identity of the two proteins and immunocytochemical studies indicate the nuclear location of the alpha-1 PI. Consistently, e.g. Ehrlich ascites tumor cells express the alpha-1 PI, and the fusion protein between the alpha-1 PI and the green fluorescent protein from Aequorea victoria shows intracellular accumulation and partly nuclear location.

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.

cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/M phase-prevalent protein.

Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (lambda CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 x 10(3) M(r)) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino-acid-transfer reactions in isolated nuclei of Ehrlich ascites tumor cells.

Nuclear enzymatic activities incorporating amino acids into acid-insoluble material were investigated with respect to their differentiation from protein biosynthesis, reaction optima, requisites and localization. The product of the reaction was analyzed with respect to its localization and nature. The nuclear activities are not inhibited by a number of inhibitors for protein biosynthesis. The reaction optima found are similar to those of other residual nuclear syntheses including the stringent dependence on ATP. All naturally occurring amino acids are utilized with different efficiencies. Their incorporation is neither cooperative nor competitive which points to individual incorporation mechanisms. Aminoacylation of tRNA may be involved because the incorporation is RNase-sensitive and aminoacylation of tRNA can be shown under the reaction conditions. The enzymatic activities are exclusively nuclear. Significant activity with unchanged characteristics is released by sonication. 70% of the radiolabel incorporated is exported across the nuclear envelope during the incubation. The residual 30% of the radiolabel is distributed without enrichment in any nuclear subfraction. The products are exclusively of polypeptide nature. Since distinct nuclear proteins (e.g. histones) which are definitely preformed in the cytoplasm by protein biosynthesis become radiolabelled by the incorporation of radiolabelled amino acids, it is evident that the incorporation takes place at preformed polypeptides. This is unequivocally proven by the incorporation of radiolabelled amino acids into exogenous proteins by means of the solubilized nuclear activities. The results indicate that the nuclear activity under investigation reflects a nuclear modification system for polypeptides which may be of similar importance as other post-translational modification systems.

Cell density dependent DNA replication in Ehrlich ascites tumour cells.

DNA replication of Ehrlich ascites tumour cells was investigated in suspensions with different cell densities by incorporation in vitro of tritiated thymidine and alkaline sucrose gradient analysis of the newly formed DNA. It is demonstrated that the incorporation of [3H]thymidine and chain growth of newly made DNA decreases with increasing cell density. The inhibition of DNA synthesis observed at high cell densities can be prevented if diffusible substances are removed by incubating the cells in dialysis tubes. This indicates that the changes in DNA synthesis are caused by diffusible inhibitors released from the tumour cells.