Alex J. van der Eb

A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155-9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [Science 230 (1985) 1350-1353] and results in a more than 10(4)-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 microgram of tumor DNA.

A Presumed DNA Helicase Encoded by ERCC-3 Is Involved in the Human Repair Disorders Xeroderma Pigmentosum and Cockayne's Syndrome

The human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 amino acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding domains and seven consecutive motifs conserved between two superfamilies of DNA and RNA helicases, strongly suggesting that it is a DNA repair helicase. ERCC-3-deficient rodent mutants phenotypically resemble the human repair syndrome xeroderma pigmentosum (XP). ERCC-3 specifically corrects the excision defect in one of the eight XP complementation groups, XP-B. The sole XP-B patient presents an exceptional conjunction of two rare repair disorders: XP and Cockaynes syndrome. This patients DNA contains a C----A transversion in the splice acceptor sequence of the last intron of the only ERCC-3 allele that is detectably expressed, leading to a 4 bp insertion in the mRNA and an inactivating frameshift in the C-terminus of the protein. Because XP is associated with predisposition to skin cancer, ERCC-3 can be considered a tumor-preventing gene.

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA

Abstract This paper reports the identification and partial characterization of a transforming activity associated with the 1 × 106-dalton HpaI E fragment of the DNA of human adenovirus type 5. This fragment represents the left-terminal 4.5% of the viral genome, which essentially corresponds to early region EIa. The transforming activity of this fragment is approximately fivefold lower than that of larger DNA fragments, as measured by focus formation in primary cultures of baby rat kidney (BRK)cells. The HpaI E-transformed cell lines were found to contain between 0.8 and 16.4 copies of fragment HpaI E per diploid amount of cell DNA and only very low concentrations of virus-specific T antigen, as detected by immunofluorescence. An analysis of some of the clones for the presence of viral RNA transcripts showed that each line contained cytoplasmic RNA hybridizing with fragment HpaI E. The size of the RNAs, however, differed from that of RN As normally transcribed from this segment of the genome. A study of the phenotypical properties of HpaI E-transformed cells showed that they can be distinguished from BRK cells transformed by larger DNA fragments by (1) a more or less fibroblastic appearance, (2) a tendency to grow in parallel orientation (a property characteristic of untransformed fibroblasts), (3) a low growth rate, (4) the inability to reach high cell densities in monolayer culture. Some of these properties changed gradually upon prolonged passaging in vitro, in particular the growth rate and, to a lesser extent, the saturation density gradually approached those characteristic for transformed cells. Apart from these differences in properties, all HpaI E-transformed cells have at least two characteristics in common with other transformed cells: they all grow as immortal cell lines and they are all aneuploid. The results indicate that the leftmost 4.5% of Ads DNA is able to convert diploid cells in a primary culture into established aneuploid cell lines which still lack several of the properties characteristic for adenovirus 5-transformed cells.

IκBα is a target for the mitogen‐activated 90 kDa ribosomal S6 kinase

The activity of transcription factor NFκB is regulated by its subcellular localization. In most cell types, NFκB is sequestered in the cytoplasm due to binding of the inhibitory protein IκBα. Stimulation of cells with a wide variety of agents results in degradation of IκBα, which allows translocation of NFκB to the nucleus. Degradation of IκBα is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen‐activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IκBα kinase. p90rsk1 phosphorylates IκBα at Ser32 and it physically associates with IκBα in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant‐negative mutant, IκBα degradation in response to mitogenic stimuli, e.g. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), is inhibited. Finally, NFκB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IκBα in response to mitogens.

Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2‐mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self‐ubiquitylation.

Comparative study of the p53-mdm2 and p53-MDMX interfaces.

Mdm2 and MDMX are two structurally related p53-binding proteins which show the highest level of sequence similarity in the N-terminal p53-binding domains. Apart from its ability to inhibit p53 mediated transcription, a feature it shares with mdm2, very little is known about the physiological functions of MDMX. It is clearly distinct from mdm2 since its expression appears not to be regulated by p53 and it cannot compensate for lack of mdm2 in early development. We present data on the structural similarity between the p53 binding pockets of mdm2 and MDMX using p53- and phage-selected peptides. From the results we conclude that our recently devised innovative approach to reverse the mdm2-mediated inhibition of p53s transactivation function in vivo would probably target MDMX as well. Strategies for selectively targeting mdm2 and MDMX are suggested and a possible mechanism for regulating the p53-mdm2/MDMX interactions by protein phosphorylation is discussed.

Localization of the E1B proteins of adenovirus 5 in transformed cells, as revealed by interaction with monoclonal antibodies

Monoclonal antibodies, one against the adenovirus type 5 E1B 55-kDa protein and one against the E1B 21-kDa protein, have been isolated and shown to recognize the same proteins as antitumor sera. Immunofluorescence studies with these monoclonal antibodies on transformed cells containing the complete adenovirus early region 1, showed that the E1B 21-kDa protein is localized in the perinuclear region. The E1B 55-kDa protein is localized in a number of different sites: a strong fluorescence is observed in a discrete body in the cytoplasm close to the nucleus, a moderate fluorescence is found in cell-cell contacts, and a weak staining in the cytoplasm. The cellular p53 antigen, which is associated with the E1B 55-kDa protein, is also found in the discrete cytoplasmic body, but not, or only in small amounts at the cell-cell contacts. However, p53 is not seen in the cytoplasm outside the discrete body, but the nucleus is weakly positive. The nature of the discrete cytoplasmic body was investigated further by electron microscopy and was found to be composed of a cluster of 8-nm filaments. The diameter of the filaments is similar to that of cytoskeletal intermediate filaments. However, staining with antibodies against the various intermediate filament proteins did not show a significant reaction with the cluster, while vimentin intermediate filaments could be demonstrated in the cells in a typical cytoskeletal pattern. It was also shown that the cluster is not composed of incorrectly aggregated tubulin.

Ultraviolet-radiation induced c-jun gene transcription: two AP-1 like binding sites mediate the response.

Abstract— In HeLa cells transcription of the c‐jun gene is activated strongly and rapidly by ultraviolet (UV) irradiation and, to a somewhat lesser extent, by treatment with phorbol ester tumor promoters. In the same cells UV and phorbol esters only marginally enhance the abundance of RNA transcribed from the jun D gene and from the gene coding for the serum response factor (which in turn acts on the UV and phorbol ester response element of the c‐fos gene). In contrast to c‐jun, jun B transcription is induced more efficiently by phorbol ester than by UV irradiation, suggesting that the members of the jun family are differently regulated. The promoter of c‐jun carries two enhancer elements resembling AP‐1 binding sites: the junl UV response element (URE‐71 TGACATCA‐64) and the jun2 URE (‐190 TTACCTCA‐183). These elements act independently in the UV induced expression of c‐jun. In the context of the complete c‐jun promoter they seem not to be required for c‐jun induction by phorbol esters. When fused to the Herpes simplex thymidine kinase promoter, however, the isolated elements mediate induction by both UV and phorbol esters. UV and phorbol ester treatment of cells increases the binding of transcription factors to both elements. Both elements bind factors different in modification or/and constitution from AP‐1, the heterodimeric transcription factor composed of c‐Fos and c‐Jun that controls the activity of the UV and phorbol ester response element (‐72 TGAGTCA‐66) of the human collagenase gene.

Transformation with specific fragments of adenovirus DNAs I. Isolation of specific fragments with transforming activity of adenovirus 2 and 5 DNA

Abstract DNA of human adenoviruses 2 and 5 was cleaved by the restriction endonucleases Hsu I, Bam HI, Hpa I, and Sma I. The resulting fragments were separated and tested for their ability to transform primary baby rat kidney (BRK) cells, using the calcium technique. Fragments with transforming activity were obtained with endos R·Eco RI (fragments A), Bam HI (fragments B of Ad2 and A of Ad5 DNA), and Hsu I (fragments G). The transforming fragments all represented the left terminal fragments of the respective restriction endonuclease cleavage products. The smallest fragment found to contain transforming activity was the Hsu I G fragment (molecular weight 1.7 · 10 6 for both Ad2 and Ad5 DNA). Transforming activity of both adeno DNAs was abolished by digestion with endos R·Hpa I and Sma I. This indicated that these enzymes cleave into an area essential for transformation. A number of cell lines transformed by restriction endonuclease fragments were established and some of their properties were studied. All adeno DNA fragment-transformed lines were found to grow to only a very low level in 0.33% agarose medium (cloning efficiency Hsu I G-transformed cells, however, was atypical and differed from the usual pattern, in that the fluorencence was largely localized in the cytoplasm. Selection of Hsu I G-transformed cells in 0.33% agarose medium resulted in cell populations containing the typical adenovirus T antigen pattern.