A. Houweling

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.

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.

Expression of region E1b of human adenoviruses in the absence of region E1a is not sufficient for complete transformation

Previous work has suggested that morphological transformation of cultured cells by human adenoviruses of subgroups A, B, and C is predominantly a function of early region 1b (E1b), and that region E1a has a role in immortalization. To test the hypothesis that region E1b is essentially responsible for the induction of the transformed phenotype the transforming activity of region E1b in the absence of region E1a was reinvestigated. In agreement with previous results, region E1b had no detectable transforming activity in primary baby rat kidney (BRK) cells nor in established rat cell lines. Since recent experimental evidence indicates that expression of E1b is blocked by a cellular factor which is inactivated by region E1a products, the regulatory signals in front of the coding sequence of region E1b were removed and replaced by the early promoter of SV40. These E1b-SV40pr plasmids had no detectable transforming activity in primary BRK cells, but they transformed normally in the presence of region E1a plasmids, demonstrating that both subregions are required for complete transformation of primary BRK cells. Transfection of the established rat cell line 3Y1 with the E1b-SV40pr plasmids did not result in complete morphological transformation either. Cotransfection of 3Y1 cells with E1b-SV40pr plasmids and pAG60 (a plasmid which harbors the kanamycin-resistance gene of Tn5) resulted in the appearance of foci of cells resistant to the antibiotic G-418. These colonies expressed the region E1b polypeptides to levels comparable to those found in cells transformed with intact region E1. Despite the presence of the E1b proteins the cells appeared essentially untransformed, in contrast to foci obtained after cotransfection of 3Y1 cells with mixtures of p5XhoI C (comprising region E1 DNA) and pAG60. These results indicate that complete transformation is a function of both regions E1a and E1b and that region E1a must have an important role in morphological transformation.

Adenovirus E1A-mediated regulation of class I MHC expression

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non‐oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I‐negative Ad12‐transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I‐positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I‐positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12‐transformed cells is due to an active switching‐off process.

Morphological transformation of human adenoviruses is determined to a large extent by gene products of region E1a.

The role of region E1a and E1b of human adenovirus (Ad) types 5 and 12 in determining the morphology of transformed colonies has been studied. Primary baby rat kidney cells were transfected with a mixture of plasmids containing Ad5 region E1a and Ad12 region E1b, or vice versa, and the morphology of the resulting transformed colonies was studied. It was found that the morphology of the colonies was correlated with the identity of the E1a region present in the cells; i.e., colonies transformed by Ad5 E1a plus Ad12 E1b resembled Ad5-transformed colonies, whereas those transformed by Ad12 E1a plus Ad5E1b resembled Ad12-transformed colonies. This suggested that the morphology of Ad-transformed cells is determined mainly by region E1a. To exclude the possibility that this phenomenon is due to an E1a-mediated and serotype-specific regulation of E1b expression and that the transformed phenotype is largely determined by region E1b, the experiments were repeated with Ad5 E1b plasmids in which the transcription regulation sequences had been replaced by the SV40 early promoter segment. In these plasmids E1b expression has become independent of region E1a. Foci of cells transformed by these E1b-SV40 promoter plasmids in the presence of either E1a of Ad5 or E1a of Ad12 again showed the same phenomenon, i.e., an Ad5-specific morphology with Ad5 E1a and Ad12-specific morphology with Ad12 E1a. Preliminary evidence showing that region E1b may regulate the concentration of E1a transcripts is discussed.

Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes

Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.

Different regulation of p53 stability in UV-irradiated normal and DNA repair deficient human cells

The stabilization of p53 protein was studied after UV exposure of normal human skin fibroblasts and cells derived from patients suffering from xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). The data show that p53 is transiently stabilized both in UV-irradiated normal and repair deficient cells. However, particularly at later times after UV irradiation, stabilization of p53 persists much longer in repair deficient XP and TTD cells than in normal cells. The stabilization of p53 was found to be dose-dependent in normal and XP cells. These results indicate that unremoved DNA damage could possibly be responsible for the induction of transient stabilization of p53.

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA.

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.

Different adenovirus E1A-controlled properties of transformed cells require different levels of E1A expression

We have transformed primary baby rat kidney cells with a plasmid containing the adenovirus(Ad)12E1 region in which the E1A promoter was replaced by the dexamethasone inducible mouse mammary tumor virus promoter. In the uninduced state the level of E1A expression is less than 10% of that in the induced state. We have investigated the effects of decreasing the levels of E1A on a number of E1A-mediated processes. First, expression of the E1B region is reduced several-fold upon reducing E1A expression. Second, a radical change in cell morphology is observed. Third, despite the decrease in E1A expression, down-regulation of the class-I major histocompatibility complex genes by Ad12E1A is not affected. These results are discussed in terms of different threshold levels of E1A expression required for various E1A-mediated processes.