Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.

Properties of rat cells transformed by DNA plasmids containing adenovirus type 12 E1 DNA or specific fragments of the E1 region: comparison of transforming frequencies.

In this paper, we describe for the first time the transformation of normal rat cells by DNA equivalent to adenovirus type 12 Early Region 1 (E1A). This DNA was 30-fold less efficient at transformation than DNA encoding the entire E1 region. Those established lines expressing a full complement of adenovirus type 12 E1 proteins were phenotypically indistinguishable from adenovirus type 12 virus-transformed cell lines. However, cell lines produced by plasmids carrying subgenomic fragments of E1 DNA and therefore not expressing E1B Mr 52,000 protein took longer to establish and produced tumors only after a protracted latent period. A Giemsa-banding study showed that adenovirus transformation can occur without disruption of the normal rat karyotype.

Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras.

The efficiency of transformation of primary human embryo retinal (HER) cells by the adenovirus E1 region (E1A + E1B) depended on the virus serotype whereas transformation by E1A alone was a rare event regardless of serotype. Activated human c-Ha-ras and N-ras genes co-operated differentially with different E1As for HER transformation but were ineffective without E1A. Ras + E1A co-transformants containing Ad 12 E1A established directly from foci, in contrast to those containing Ad 2 or Ad 5 E1A. A spectrum of activated ras gene expression was found in stable co-transformants with mRNA and protein levels being lower in Ad 12 E1A + N-ras than Ad 2 E1A + N-ras cell lines. Down regulation of E1A transcription in the absence of E1B was found in Ad 2 E1A + Ha-ras transformants only but E1A protein levels were similar to those in Ad 2 E1A + N-ras or Ad 5 E1A + E1B cell lines. HER cell transformants which contained Ad 12 E1A were more tumourigenic than those which contained the Ad 2 or Ad 5 E1A. This unique transformation system shows that stable malignant transformation of primary human cells in vitro is a complex process requiring the combined activities of two or more types of genes.

The expression of Ad12 E1B 19K protein on the surface of adenovirus transformed and infected human cells.

Using radioimmunoassays we have confirmed earlier reports that the adenovirus 12 E1B 19K protein is expressed on the surface of transformed and infected human cells. Immunoprecipitation and radioimmunoassay of Ad 12 infected primary human embryo kidney cells demonstrated a considerable time lag in the translocation of the 19K protein to the external membrane after its expression within the cell. We have used antisera raised against synthetic octapeptides to detect and map the orientation of the polypeptide in the plasma membrane. The data obtained suggest that the C-terminus of the protein is exposed on the outside of the cell whereas the N-terminus is free in the cytoplasm. The predicted secondary structure of the 19K protein demonstrates a hydrophobic region which is highly conserved amongst Ad2, 5, 7 and 12 (amino acids 83-100 in Ad12) and we conclude that this forms the transmembrane domain. These observations are discussed in relation to the known functions of the Ad12 E1B 19K protein.

Rearrangement of the same chromosome regions in different SV40 transformed human skin keratinocyte lines is associated with tumourigenicity.

Twelve different human keratinocyte strains were transformed with recombinant plasmid pSV6-1 which contained an origin defective SV40 genome. When injected into athymic nude mice lines produced either squamous cell carcinomas (SCC) in all animals, SCC in some animals and epidermal cysts in others, or epidermal cysts only in all the animals. The tumourigenic capacity of the lines could be correlated with the chromosomal changes present initially in the transformed cells. Lines which produced SCC in all the animals within a short period of time all showed simultaneous loss of part of chromosomes 3p, 8p and 11p in one homologue. Lines which were not tumourigenic did not show these simultaneously appearing rearrangements. These specific rearrangements are acquired in vitro and the time taken for a recognisable tumour to appear is related to the proportion of such cells in the line. The rearrangement of the same chromosome regions in different tumourigenic cell lines suggests that genes in these regions are important in the development of squamous cell carcinoma, possibly by loss of heterozygosity, at particular loci.

Purification and characterisation of the protein encoded by the activated human N-ras gene and its membrane localisation.

Using a transformed human embryo retinal cell line (Ad 2 E1A + N-ras HER 313A), which expresses activated N-ras p21 at a high level, we have examined various properties of the protein. Immunoprecipitated p21 has covalently bound lipid attached to it through an alkali-labile thioester bond. This incorporation of fatty acid into the protein proceeds in vitro and probably in vivo via a palmitoyl-CoA intermediate and is catalysed by a crude microsomal preparation. A novel purification procedure has been developed for the protein based on its solubility in high concentrations of ethanol. Residual protein impurities were removed by gel filtration in the presence of detergent. Using a membrane preparation from Ad 2 E1A + N-ras HER 313A cells, we have shown that N-ras p21 is firmly anchored in the cell membrane and is not removed by extraction with salts, chelating agents or reducing agents, but is only solubilised by detergents at high concentrations. Exposure of cell membrane preparations and purified N-ras p21 to proteolytic enzymes gives rise to similar degradation patterns. Based on these observations and the known amino acid sequence of p21, it is concluded that attachment to the cell membrane is through the lipid at the C-terminus and not through the incorporation of the polypeptide chain into the lipid bilayer. These results are discussed in relation to the hypothesis that the mode of action of p21 is analogous to that of G proteins.

The binding of guanine nucleotide to N-ras p21--a phosphorous and proton magnetic resonance study.

One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.

Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells.

The adenovirus early region 1B (Ad E1B) genes have no transforming capability of their own but markedly increase the transformation frequency of Ad E1A following co-transfection into mammalian cells. The larger E1B proteins of both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptionally activate other genes. We have previously demonstrated that synthetic peptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro. In the work presented here we have examined the effects of complex dissociation on Ad E1-transformed human cells. It has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-transformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocated to the nucleus. In Ad12 E1-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized, 'flecked' distribution to one in which nuclear staining was homogeneous and diffuse. Peptides added to either Ad5 E1 or Ad12 E1 transformed cells resulted in the release of transcriptionally active p53. Interestingly, the level of p53 then fell presumably as a result of proteasomal action - this was probably a reflection of the short half-life of 'free' (i.e. dissociated) p53 compared to that of the bound protein. Free p53 did not cause apoptosis in target cells probably due to the presence of the smaller (19K) E1B proteins. However, addition of peptide leads to a significant reduction in cell growth rate. We have further demonstrated that a significant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger EIB protein during transformation is considered in view of these data.

Cellular localization and T antigen binding of the retinoblastoma protein.

Mutation of the retinoblastoma (Rb) gene is found frequently in human sarcomas, lung, bladder and breast carcinomas and is the molecular basis for hereditary predisposition to retinoblastoma. The Rb protein is a nuclear phosphoprotein that is differentially phosphorylated during the cell cycle. Its precise function is unknown but it has been suggested that it may act as a transcriptional regulator or as a regulator of cellular DNA synthesis. The Rb protein forms specific complexes with the oncogenes of three different groups of DNA tumour viruses. We have prepared a new monoclonal antibody to the Rb protein and used it to establish sensitive immunoassays for Rb complexed to T antigen. In SV40-infected and transformed cells these assays showed that Rb enters a trimolecular complex containing p53, Rb and T. A large panel of human tumour cell lines was tested for expression, cellular localization and T-binding activity of Rb using the new antibody.

Definition of a major p53 binding site on Ad2E1B58K protein and a possible nuclear localization signal on the Ad12E1B54K protein.

Previous studies have established that adenovirus 2/5 early region 1B (Ad E1B) 58K protein binds p53 strongly and co-localizes with it to cytoplasmic dense bodies whilst the homologous Ad12E1B54K protein binds only weakly and co-localizes primarily to the nucleus in Ad12E1 transformed cells. We have used these properties of the E1B proteins from different viral serotypes to map the p53 binding site on the Ad2/5 protein. A set of chimaeric genes was constructed containing different proportions of the Ad12 and Ad2E1B DNA. These, together with Ad12E1A and E1B19K DNA, were transfected into baby rat kidney cells and transformed lines isolated. From an examination of the properties of these Ad12/Ad2E1B fusion proteins in co-immunoprecipitation and subcellular localization experiments it has been concluded that the p53 binding site on Ad2E1B58K protein lies between amino acids 216 and 235 and that the homologous region on Ad12E1B54K protein also binds p53. In addition, a unique nuclear localization signal is located on Ad12E1B54K between residues 228 and 239. We suggest that primary structure differences in these regions of the Ad2 and Ad12E1B proteins are responsible for the different subcellular localizations in AdE1 transformants.

Sensitivity to ionising radiation of transformed human cells containing mutant ras genes.

Measurement of colony forming ability following exposure to gamma-rays has been performed on human retinoblasts transformed with either adenovirus 5 or 12 early region 1 DNA, adenovirus early region 1A plus activated N- or H-ras DNA or SV40 DNA. In contrast to recently reported results (M.D. Sklar, 1988, Science, 239, 645-647), we found no general correlation between transformation with activated ras and increased radiation resistance. Similarly, there was no correlation between D0 values and the level of expression of ras p21 in transformed human retinoblasts as determined by liquid competition assay. Indeed, cell lines with very similar D0 values had ras contents varying by up to one hundred fold. Cell lines transformed with SV40 DNA were generally less sensitive to ionising radiation than adenovirus and/or ras transformants, but even so the variation in sensitivity within these encompassed the whole spectrum of values obtained for the ras transformants. It may be interesting to note, however, that two out of the three ras transformants which were least sensitive to gamma-rays were cell lines expressing the highest levels of p21.

Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.

We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.

Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome.

Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.

The expression of the retinoblastoma gene product Rb1 in primary and adenovirus-transformed human cells.

Polyclonal antibodies to the human retinoblastoma gene product (Rb1) have been produced in rats by immunisation with a fusion protein comprising part of Rb1 together with the E. coli beta-Gal sequence. We have used these antibodies in Western blotting studies to screen a number of human foetal tissues and organs and found approximately similar levels of expression of Rb1 in all of them. The protein seems to be somewhat more abundant in some cell lines produced by transfection of human embryo retinal (HER) cells with adenovirus 12 early region 1 (Ad 12 E1), Ad 5 E1, Ad 2 E1A + mutant N-ras or SV40 DNA. Using co-immunoprecipitation followed by Western blotting we have shown that the Rb1 protein binds to Ad 12 E1A 266 and 235 amino acid proteins. This interaction is ionic strength dependent but is unaffected by non-ionic detergent up to a concentration of at least 1%. In Ad 12 infected human cells it appears that less E1A is bound to Rb1 than in the transformants. These results are discussed in view of the known similarities and differences between the amino acid sequences of Ad 12 and Ad 5 E1A proteins.

The expression of MDM2 and other p53-regulated proteins in the tissues of the developing rat.

The expression of MDM2 was monitored in tissues taken from rats at different stages of development. Two major protein species of 90K and 130K were identified, although expression was not the same in all tissues. In most the level of the larger protein tended to decrease as the foetus developed, being relatively scarce after birth. The expression of the 90K product (usually considered to be the major MDM2 protein), however, increased as the animals developed, being most abundant in adult tissues, suggesting that the two proteins play quite different roles during development. p53 expression mirrored that of the smaller MDM2 protein in most, but not all, tissues. This is consistent with previous suggestions made on the basis of in vitro studies that expression of the latter protein is regulated by p53. In addition, the level of two other p53-regulated proteins, Bcl-2 and Bax, were examined. In most tissues expression of both proteins decreased as the foetus developed, being virtually undetectable in adults. These data strongly suggest that factors other than p53 exert the major influence over Bcl-2 and Bax expression, although levels of the 90K MDM2 protein are more likely to be regulated by p53. However, it was noted that the levels of MDM2 in the tissues of p53-null mice were comparable to those seen in the normal rat.

Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein.

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K(i) of 1.04, 1.34 and 0.52 microM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K(i) of 332 and 56 microM, respectively) whilst the Hairy peptide bound much more strongly (K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.

Purification of a serum factor which reverses dibutyryl cAMP induced differentiation.

We have previously shown [Grabham et al. (1988) Expt. Eye Res. 47, 123-133] that the adenovirus 12 transformed human retinoblast cell line (Ad 12 HER 10), like a number of other cell types of neuroepithelial origin, can be induced to differentiate in response to exposure to dibutyryl cAMP, and that this differentiation can be rapidly reversed by foetal calf serum. We present data here to show that a single protein, which we have termed differentiation reversal factor (DRF) and have isolated from serum, is responsible for this activity. Following reversal by DRF the growth rate of these cells was shown to be stimulated in serum-free medium. Using ammonium sulphate fractionation, gel filtration chromatography (Ultrogel AcA44), anion exchange chromatography (DEAE cellulose) and preparative gel electrophoresis, DRF has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis in the presence and absence of SDS. The protein has a mol. wt of 72,000 and appears to exist in vivo as a monomer. The concentration of DRF in serum is in the range 100-500 micrograms/ml and is capable of reversing cAMP-induced differentiation of various primary human neuroepithelial cells at physiological concentrations.

Acylation of adenovirus type 12 early region 1b 18-kDa protein. Further evidence for its localisation in the cell membrane.

The 18-kDa E1b protein in Ad 12-transformed rat cells and in Ad 12-infected human cells binds lipid strongly. The lipid is not removed by boiling in the presence of SDS or by extraction with methanol/chloroform. It is, however, dissociated from the protein by treatment with methanolic KOH suggesting that attachment is through an ester linkage. The acylated 18-kDa protein is detected only in the membrane fraction. Labelling cell surface proteins on Ad 12-transformed cells with [125I]iodosulphanilic acid shows that some of the Ad 12 18-kDa E1b protein is present on the outside of the cell. It is concluded that this protein is responsible for cell surface T-antigen activity.

Modulation of Human Neurite Outgrowth by Serine Proteases: A Comparison of the Interaction of Thrombin and Prothrombin with Glia-Derived Nexin.

Neurite outgrowth from cells of neuroepithelial origin is under the reciprocal control of thrombin and the thrombin inhibitor-glia-derived nexin (GDN). The neurite retraction activity of thrombin is blocked when GDN complexes with the enzyme and inhibits its proteolytic activity. However, we have previously shown that enzymically inactive proenzyme is also capable of inducing neurite retraction. We present evidence here to show that GDN does not bind to prothrombin in solution. When a mixture of prothrombin and GDN is subjected to either polyacrylamide gel electrophoresis or immunoprecipitation, a stable complex cannot be detected. This is in direct contrast to thrombin, which exhibits stable complexes with GDN under both conditions. At the cell surface, however, GDN is able to inhibit the biological activity of prothrombin. When a mixture of proenzyme and inhibitor is applied to previously differentiated transformed retinoblasts (Ad12 HER10), the ability of prothrombin to induce neurite retraction is blocked. Furthermore, following 1 h exposure to Ad12 HER10 cells, a solution of prothrombin was found to contain half the potential enzyme activity as detected by chromogenic assay. These results have been interpreted as evidence for the ability of neuronal cells to cleave prothrombin and subsequently release activated enzyme.

Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes.

Few human cell systems have been described in which a number of different genes induce transformation. The present investigation reports on our studies using primary human embryo retinoblasts as a model system to monitor transformation and the subsequent behaviour of individual transformants in terms of establishment, the frequency of immortalization and tumourigenic potential. SV40, Adenovirus E1 and E1A, and combinations of Adenovirus E1A and activated H-ras or N-ras were examined as transforming agents. Considerable differences were observed in the ability of these genes to transform human cells, to induce immortal lines and to produce cell lines with a tumourigenic phenotype. Activated ras genes were non-transforming in this system and the degree of complementation with adenovirus E1As in transformation experiments was dependent on both the adenovirus serotype and the ras gene used. The development of tumourigenic cell lines required the expression of more than one oncogene and additional genetic events were required in some in some instances before immortal cell lines were obtained. These findings contribute to the concept that the development of cancer is a multi-step process.