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The hSNF5 subunit of human SWI/SNF ATP-dependent chromatin remodeling complexes is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). Here, we report that loss of hSNF5 function in MRT-derived cells leads to polyploidization and chromosomal instability. Re-expression of hSNF5 restored the coupling between cell cycle progression and ploidy checkpoints. In contrast, cancer-associated hSNF5 mutants harboring specific single amino acid substitutions exacerbated poly- and aneuploidization, due to abrogated chromosome segregation. We found that hSNF5 activates the mitotic checkpoint through the p16INK4a-cyclinD/CDK4-pRb-E2F pathway. These results establish that poly- and aneuploidy of tumor cells can result from mutations in a chromatin remodeler.

The hSNF5 chromatin-remodeling factor is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). A number of studies have shown that hSNF5 re-expression blocks MRT cell proliferation. However, the pathway through which hSNF5 acts remains unknown. To address this question, we generated MRT-derived cell lines in which restoration of hSNF5 expression leads to an accumulation in G0/G1, induces cellular senescence and increased apoptosis. Following hSNF5 expression, we observed transcriptional activation of the tumor suppressor p16INK4a but not of p14ARF, repression of several cyclins and CD44, a cell surface glycoprotein implicated in metastasis. Chromatin immunoprecipitations indicated that hSNF5 activates p16INK4a transcription and CD44 down-regulation by mediating recruitment of the SWI/SNF complex. Thus, hSNF5 acts as a dualistic co-regulator that, depending on the promoter context, can either mediate activation or repression. Three lines of evidence established that p16INK4a is an essential effector of hSNF5-induced cell cycle arrest. 1) Overexpression of p16INK4a mimics the effect of hSNF5 induction and leads to cellular senescence. 2) Expression of a p16INK4a-insensitive form of CDK4 obstructs hSNF5-induced cell cycle arrest. 3) Inhibition of p16INK4a activation by siRNA blocks hSNF5-mediated cellular senescence. Collectively, these results indicate that in human MRT cells, the p16INK4a/pRb, rather than the p14ARF/p53 pathway, mediates hSNF5-induced cellular senescence.

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.

Currently, the preferred host for the production of early region-1 (E1)-deleted recombinant adenoviruses (rAdV) is cell line 293, which was generated by transformation of human embryonic kidney cells by sheared adenovirus 5 (Ad5) DNA. To develop alternative hosts for the production of rAdV, we generated adenovirus-transformed human cell lines by transformation of human embryonic retinoblasts (HER) with a plasmid containing base pairs 79-5789 of the Ad5 genome. One of the established HER cell lines, which we called 911, exhibited favorable growth characteristics and was chosen for further study. This cell line is demonstrated to have several characteristics in common with the well-known 293 cell line: The 911 cell line is highly transfectable, and exhibits similar frequencies of homologous recombination. However, it has additional characteristics that make it a useful alternative for 293. The 911 cells perform particularly well in plaque assays. Upon infection with E1-deleted adenoviruses, plaques become apparent in monolayers of 911 cells already after 3-4 days versus 4-10 days in monolayers of 293 cells, thereby reducing the time required for quantitative plaque assays. Furthermore, yields of E1-deleted adenovirus vectors up to three times as high as those achieved with 293 cells can be obtained with 911 cells. Finally, the Ad5-DNA content of the 911 cell line is completely known. These features make the 911 cell line a useful alternative for the construction, propagation, and titration of E1-deleted recombinant adenoviruses.

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.

The hSNF5 chromatin-remodeling factor is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). A number of studies have shown that hSNF5 re-expression blocks MRT cell proliferation. However, the pathway through which hSNF5 acts remains unknown. To address this question, we generated MRT-derived cell lines in which restoration of hSNF5 expression leads to an accumulation in G(0)/G(1), induces cellular senescence and increased apoptosis. Following hSNF5 expression, we observed transcriptional activation of the tumor suppressor p16(INK4a) but not of p14(ARF), repression of several cyclins and CD44, a cell surface glycoprotein implicated in metastasis. Chromatin immunoprecipitations indicated that hSNF5 activates p16(INK4a) transcription and CD44 down-regulation by mediating recruitment of the SWI/SNF complex. Thus, hSNF5 acts as a dualistic co-regulator that, depending on the promoter context, can either mediate activation or repression. Three lines of evidence established that p16(INK4a) is an essential effector of hSNF5-induced cell cycle arrest. 1) Overexpression of p16(INK4a) mimics the effect of hSNF5 induction and leads to cellular senescence. 2) Expression of a p16(INK4a)-insensitive form of CDK4 obstructs hSNF5-induced cell cycle arrest. 3) Inhibition of p16(INK4a) activation by siRNA blocks hSNF5-mediated cellular senescence. Collectively, these results indicate that in human MRT cells, the p16(INK4a)/pRb, rather than the p14(ARF)/p53 pathway, mediates hSNF5-induced cellular senescence.

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.

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.

By means of immunofluorescence using two different fluorochromes antigens of B-type and C-type oncornavirus could be detected simulataneously in mammary tumour cultures. No mutual interference between both kinds of viruses could be detected.

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.

To avoid the chemical purification of mastomys IgM, a reliable alternative method for producing a specific anti-mastomys IgM antiserum was developed. It employs the strong cross-reactivity of antisera against mouse serum proteins with mastomys serum proteins. By the immunoelectrophoresis technique, precipitates of mastomys IgM and cross-reacting rabbit anti-mouse IgM were obtained. A rabbit was immunized with these precipitates. Activity against the light chains was removed from the antiserum by passage over an immunoadsorbent column of mastomys IgG. The adsorbed antiserum was found to be specific for determinants on mastomys IgM.

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.

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 endo's 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 endo's 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.

Rat cells transformed by the highly oncogenic adenovirus 12 lack at least two cellular proteins which are present in cells transformed by the non-oncogenic adenovirus 5 and in untransformed cells. One protein has been identified as the heavy chain of the rat class I major histocompatibility complex. This finding may explain the difference in oncogenicity between adenoviral species.

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment Hsu I 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 32 P-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 32 P-labeled adenovirus 5 DNA with endo R· Hsu I, or by digestion of 32 P-labeled fragment Hsu I G of adeno 5 DNA with endo R· Hpa I. All five transformed lines were found to contain DNA sequences homologous to 75–80% of Ad5 fragment Hsu I 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.