Nicole Fossar

Increased expression of cytokeratin and ferritin-H genes in tumorigenic clones of the SW 613-S human colon carcinoma cell line☆

Subclones of the SW 613-S human colon carcinoma cell line differ by their ability to induce tumors in nude mice and by their level of amplification of the c-myc gene. Clones with a high level of amplification are tumorigenic in nude mice whereas those with a low level are not. Genes overexpressed in the tumorigenic clones as compared to the nontumorigenic ones were searched by differential screening of a cDNA library. Two cDNA clones corresponding to cytokeratin K18 and ferritin-H chain were isolated. The steady state level of the corresponding mRNAs is higher in cells of all tumorigenic clones. The level of cytokeratin K8 mRNA, the specific partner of cytokeratin K18 in intermediate filaments of epithelial cells, is also elevated in these cells. For all three genes, this is mainly due to an increase in the transcription rate, as shown by a nuclear run-on assay. Immunoblotting experiments showed that cytokeratins K8, K18, and K19 are more abundant in cells of tumorigenic clones. The mRNA of the other subunit of apo-ferritin (ferritin-L chain) is expressed at the same level in both types of clones. The mRNAs of cytokeratins K18 and K8 and of ferritin-H chain are also overexpressed in cells of nontumorigenic clones which have acquired a tumorigenic phenotype after transfection of c-myc gene copies.

An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line.

Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.

Structure of four amplified DNA novel joints

The structures of four novel joints present in the amplified DNA of a Syrian hamster cell line highly resistant to N-(phosphonacetyl)-l-aspartate were analyzed. Novel joints J1, J2, and J4 were formed by recombination between two regions of wild-type DNA, whereas joint J3 is the end point of an inverted duplication. A fraction of the J3 copies displays a cruciform structure in the purified genomic DNA. The formation of J1 and J2 apparently involved a simple breakage and joining of the two wild-type sequences, whereas extra nucleotides are present at the junction point of J3 and J4. The two regions of the wild-type DNA which have recombined to form J1, J2, and J4 show few sequence similarities, indicating that these joints probably resulted from nonhomologous recombination. AT-rich regions are present in the vicinity of the breakpoint for the four joints and eight of 10 crossover points could be associated with putative topoisomerase I cleavage sites. Our results indicate that different types of novel joints are present in the amplified DNA of this cell line, which was isolated after several steps of selection.

Endonucleolytic DNase in oncorna viruses: role of mycoplasma.

Abstract The DNA-directed DNA polymerase activity of avian myeloblastosis virus and Rauscher leukemia virus was not enhanced by SV40 DNA, whereas that of murine sarcoma virus preparations was. The latter stimulation is ascribed to an endonucleolytic DNase which occurs in a mycoplasma contaminant. This can be removed by differential velocity centrifugation. Deoxyribonuclease activity is not a property of oncorna viruses, and the possibility is raised that other nonpolymerase enzymatic activities associated with these viruses may be contaminants.

High frequency trans-splicing in a cell line producing spliced and polyadenylated RNA polymerase I transcripts from an rDNA-myc chimeric gene

The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3′ end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5′ exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells.

Deregulated Expression of the Keratin 18 Gene in Human Colon Carcinoma Cells

The keratin 18 (K18) gene is expressed at a normal level in cells of nontumorigenic clones derived from the SW613-S human colon carcinoma cell line, but is overexpressed in cells of tumorigenic clones. A high level of expression was also found in the cells from 10 of 15 other human colon carcinoma cell lines. The expression of the gene is downregulated in differentiating Caco-2 cells, resulting in a normal expression level. Determination of K18 mRNA half-life in growing and confluent Caco-2 cells indicated that this downregulation does not take place at a posttranscriptional level. The density of RNA polymerase molecules on the K18 gene, as measured in nuclear run-on experiments, is the same in growing and confluent Caco-2 cells, but the rate of synthesis of K18 transcripts in confluent Caco-2 cells, as determined by in vivo pulse-labeling, is 35% of that in growing cells. Nuclear run-on experiments carried out with nuclei prepared from growing or confluent Caco-2 cells treated with 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole (DRB) indicated that a reduction in both the initiation and elongation rates of RNA polymerase molecules occurs on the K18 gene in confluent Caco-2 cells. This leads to a decreased rate of K18 transcript production with no reduction in the polymerase density on the gene. Evidence is provided that the mechanisms responsible for the differential expression of the K18 gene between tumorigenic and nontumorigenic SW613-S cells and between growing and differentiating Caco-2 cells share some similarities.

Poly (A) polymerase activity in L cells following encephalomyocarditis virus infection.

Poly (A) polymerase activity has been measured in crude cytoplasmic extracts of mouse L cells infected with encephalomyocarditis (EMC) virus. After infection there is first a decrease in enzyme activity followed by an increase which itself precedes detectable virus RNA and protein synthesis. The activity of the enzyme then declines before the release of mature virions and cell death take place. The early inhibition of poly (A) polymerase activity is correlated with the virus-induced shut-off of cellular protein synthesis but it is not due to inhibition of the synthesis of cellular enzyme and occurs in the absence of virus replication. The poly (A) polymerase is not synthesized after infection and modification of its activity can be reversed late in the virus cycle. These results indicate that host poly (A) polymerase activity can be regulated by the virus and further show that there is a correlation between the modification of poly (A) polymerase activity and the biosynthesis of poly (A).

Binding of proteins, including pp60src, to activated CH-Sepharose 4B

Activated CH-Sepharose 4B and protein A Sepharose CL-4B can bind, selectively and non-specifically, polypeptides from chick embryo cells. The major polypeptides bound have apparent molecular masses of 57–60 kDa and 47–49 kDa and cannot be eluted by extensive washing with buffers containing detergents. One of the 57–60 kDa polypeptides was identified by immunoblotting as the transforming protein of Rous Sarcoma Virus (RSV), pp60src. This polypeptide could be removed from the solid phase immunoabsorbant with 60% dimethylsulfoxide, but not with 2% SDS, 5% β-mercaptoethanol, 1 M NaCl or 0.1% Tween 20.