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The goal of this study was to determine the effect of cement mixing time and, hence, cement viscosity on the biomechanical behavior of femoral fracture fixation.

We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common α-subunit (α-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12S AdE1A inhibited α-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the α-subunit promoter. Akin to 12S AdE1A, exogenous p53 expression repressed α-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit α-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced α-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of α-subunit activity.

Human papillomavirus (HPV) antigens are expressed in epithelial cells at different stages of differentiation, and this may affect how they are handled by the immune system. We assessed the relative immunogenicities of four different HPV type 1 proteins: E6 and E7, which are made early in basal or parabasal cells; E4, which is made suprabasally in differentiating cells; and L1, a late protein which appears in the highly differentiated upper spinous layers. Pools of 15-mer peptides covering the primary sequences of all four proteins were used to screen 15 normal donors in enzyme-linked immunospot assays of gamma interferon release for both CD4(+)- and CD8(+)-T-cell reactivities. CD8(+)-T-cell responses were detected to the L1 protein in 7 of the 15 samples examined. No responses to E6, E7, or E4 were detected. CD4(+)-T-cell reactivities were again detected in 7 of the 15 donors. A broader spectrum of responses to E6 (three of seven), E4 (six of seven), and L1 (three of seven) was apparent, but there was no reactivity to E7. The predominant CD4(+) response was to E4. Reactivities were seen in some cases to corresponding regions on other common HPV types but were probably due to a multiple infection rather than to a cross-reaction. Antibodies to HPV1 virus-like particles were detected in 12 of the 15 (80%) donors, but antibody status did not correlate with T-cell reactivity. The differences in the relative immunogenicities of the four proteins revealed in this study are discussed in relation to how they may be processed and presented to the immune system by differentiating epithelial cells.

The interaction between the C-terminal binding protein 1 (CtBP-1) and purified Ad12 E1A protein has been examined through the use of a combination of biophysical techniques. A fragment equivalent to the 77 C-terminal amino acids of Ad12 E1A (Ad12 77-a.a. E1A) was generated by limited proteolysis of Ad12 266-a.a. E1A at Phe187and/or Tyr189using chymotrypsin. The impact of deletion of the 189 N-terminal amino acids from E1A on the equilibrium dissociation constant Kdfor binding to CtBP was assessed using ELISA in vitrobinding assays and intrinsic fluorescence spectroscopy. Values of Kdof 4.0 and 38 nM were determined for full-length and truncated forms of E1A, respectively. Circular dichroism spectroscopic studies revealed that the conformation adopted by these polypeptides is dependent on the surrounding environment, which is predominately randomly folded when free in solution, but adopting a more ordered α-helical secondary structure in the presence of trifluoroethanol. Using nuclear magnetic resonance (NMR) spectroscopy to examine the interaction between Ad E1A and CtBP it was observed that the chemical shift positions of individual backbone amide nitrogen atoms were well resolved in 15N-1H-HSQC NMR spectra performed on samples of isotopically 15N-labeled Ad12 77-a.a. E1A. In the presence of CtBP, signals of backbone amide nitrogen atoms displayed increased linewidth consistent with an increase in molecular mass upon binding CtBP. In addition, some signals that have been attributed to Val254/256and Leu259, and reside within the binding site for CtBP on E1A, are shifted in the 15N- and/or 1H-dimensions, defining specific contacts between E1A and CtBP. These data suggest that structural determinants in the C-terminal PXDLS binding motif in the rest of exon 2 and in exon 1 all contribute to optimizing the conformation of the binding site on Ad12 E1A for CtBP. However, no interaction was observed between CtBP and truncated Ad12 E1A, which no longer contained the C-terminal binding motif.

Previous detailed mutational analysis has shown that the binding site on adenovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is located toward the N terminus of conserved region 3 (CR3). Here we demonstrate that synthetic peptides of between 15 and 22 amino acids, identical to amino acid sequences of CR3 present in the larger Ad5 E1A (13 S product) and in both the Ad12 E1A (13 and 12 S products) proteins that lie N-terminal to the zinc finger motif, can disrupt binding of E1A to TBP. These findings suggest that the peptides are biologically active in terms of interacting with TBP and must therefore comprise some, if not all, of the TBP binding site on E1A. The interaction between Ad12 E1A and TBP was confirmed by direct co-precipitation experiments. In 1H NMR studies of CR3 peptides, regular patterns of NOEs were observed from which their conformational preferences in aqueous solution were determined. Both Ad5 and Ad12 peptides were shown to contain regions of helical backbone structure in 50% trifluoroethanol. In each case, the type and intensities of NOE cross-peaks observed correlated best to α-helical turns. These helices are more extensive in larger peptides and extend from Glu141to Val147and from Arg144to Pro152in the full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure of a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-length protein that abolishes TBP binding was examined. No significant differences between the substituted and wild type peptides were observed, suggesting that this substitution in the intact protein may cause disruption of global rather than local structures.

ABSTRACTRecent reports suggest that an early region 1B (E1B) 55,000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV‐1. Five of these were found to recognize denaturation‐resistant epitopes as determined by Western blotting–and their binding sites were identified by determining their reactivity against a panel of bacterial E4–beta‐galactosidase fusion proteins which contained progressive deletions at the C‐terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope‐defined mAbs, along with anti‐peptide antibodies raised against putative N‐ and C‐terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV‐1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5′ end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N‐terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 × 10⁵. After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus — one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 × 10⁶). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component. Copyright 1995, 1999 Academic Press

The expression of p53 in a large panel of adenovirus (Ad) 2/5- and 12-transformed human, rat, and mouse cells has been examined. In all cases, in the absence of the larger Ad E1B protein, the level of p53 is very low. In human and rat cells when the Ad 12 E1B 54K polypeptide is expressed, p53 is much more abundant, although this is not the case in Ad 12 E1-transformed mouse cells. We conclude that expression of p53 is determined by virus serotype, host cell type, and viral proteins expressed. p53 in Ad 12 E1-transformed human cells is wild type but has an extended half-life. Stabilization is not through protein-protein interaction with the Ad E1B protein. The level of expression of c-Myc is also elevated in Ad-transformed human cells but this does not correlate with the presence of the E1B protein or with p53. However, Northern blot analysis indicates a direct correlation between mRNA and protein levels. We conclude that c-Myc is regulated at the transcriptional level, whereas p53 is regulated at the post-translational level in adenovirus transformants. Copyright 1993, 1999 Academic Press

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.

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.

Human papillomavirus (HPV) type 1 (HPV 1) is associated with benign cutaneous warts and HPV type 16 (HPV 16) with mucosal epithelial lesions that can progress to invasive carcinoma. The primary structure of the HPV E4 proteins is not highly conserved between types and their role in the viral life cycle is still unknown. A large panel of Simian virus 40 (SV40)-transformed human and monkey epithelial and fibroblast cell lines were infected with recombinant SV40/HPV 1 E4 or SV40/HPV 16 E4 viruses and the expression of the viral proteins was analyzed by indirect immunofluorescence. Both HPV 1 and HPV 16 E4 proteins formed extensive and organized filamentous cytoplasmic networks that co-localized with the cytokeratin intermediate filaments. However, only HPV 16 E4 induced the collapse of the cytokeratin filaments. Furthermore, when both virus type E4 proteins were expressed within the same cell the collapse of the HPV 16 E4 filaments did not induce the collapse of the HPV 1 E4 network. Similar E4 filamentous structures were also observed in the cytoplasm of cells of the parabasal layer of an HPV 1-induced experimental wart. The HPV 16 E4 protein formed cytoplasmic networks in all SV4O-transformed cell lines examined, but HPV 1 E4 only formed filamentous networks in human keratinocytes and in a monkey stomach epithelial cell line. In keratinocyte cells HPV 1 E4 species of 16, 17, 32, and 34 kDa were expressed, while in Cos-1 cells (in which no E4 networks are formed) only the 17 and 34 kDa polypeptides were found. The specific behavior of E4 proteins of cutaneous and mucosal HPVs expressed in cultured cells may suggest that these viral proteins have evolved to perform a similar function at different epithelial sites. Copyright 1993, 1999 Academic Press

Study of the human papillomavirus (HPV) E4 gene product has focused largely on HPV type 1 (HPV 1) primarily because of the large quantities of protein that can be purified from HPV 1-induced warts. We have extended the characterization of the HPV 1 E4 protein and, in this study, have shown that protein purified from clinical material and a heterologous expression system contains the divalent metal ion zinc. Furthermore, using a [65Zn]Cl2dot-blot assay, we have shown that this binding is specific for zinc and those divalent cations that are known to structurally substitute zinc in metalloproteins. Mutational analysis has demonstrated that histidine amino acids (residues 56, 86, and 121), but not the cysteine residue (115), are essential for the zinc-binding activity of the E4 protein. Two assayable functions of E4 are dimerization and the formation of E4/cytokeratin structures in cultured cells; however, neither activity is abrogated by the loss of zinc binding.

The expression of p53 in human cells infected with wild-type (wt) and mutant adenoviruses has been examined. With wt Ad5 and Ad12, and Ad12 viruses carrying lesions in the E1A or the 19K E1B genes, there was a pronounced decrease in level of p53 during the course of infection. However, when cells were infected with mutant viruses which did not express the larger E1B proteins (Ad12 dl620 and in602 and Ad5 dl338 and pm381) the concentration of p53 increased markedly to levels comparable to those seen in adenovirus transformed cells. This increase in level of p53 correlated closely with the advent of E1A expression. Infection with Ad5 dl355 (which carries a lesion in the E4 gene) also resulted in an increase in p53 expression. We have concluded that these results can be explained on the basis of the known ability of EtA to stabilize p53 and of the E1B 58K:E434K protein complex to regulate mRNA metabolism during viral infection, although large increases in expression of p53 or any other cellular proteins following infection with these viruses have not previously been reported. It is suggested that the high concentrations of p53 could explain the inability of 54K and 58K negative mutants to transform cells in culture. In cells infected with dl355 both the Ad5 E1B 58K protein and p53 were located in the nucleus. It was shown by coimmunoprecipitation experiments that these proteins formed a complex which was stable in the presence of high concentrations of NaCl. The interaction of the Ad12 E1B 54K protein and p53 has also been demonstrated in Ad12 E1-transformed cells by immunoprecipitation experiments. These data, taken in conjunction with previous results, have suggested that increased expression of p53 is unrelated to complex formation with the larger Ad E1B proteins. Copyright 1994, 1999 Academic Press

By Southern blotting analysis, six adenovirus type 12 (Ad-12)-transformed rat embryo brain cell lines and one Ad-12-transformed rat liver epithelial line were shown to contain all or nearly all the Ad-12 genome. Another Ad-12 rat liver epithelial cell line contained a repeating structure consisting of only the left-hand 16% of the Ad-12 genome. Three Ad-12-specified proteins (molecular weights, 52,000, 41,000, and 18,000) were found by immunoprecipitation to be common to all of these cell lines. Two rat liver epithelial lines, produced from an Ad-12-infected culture and previously shown to be T-antigen negative by immunofluorescence, contained no detectable Ad-12 genome or Ad-12-specified proteins. Although some of the rat embryo brain transformants had been shown previously to express "late" Ad-12 mRNA, no Ad-12 structural proteins were found to be produced by these cell lines.

The level of p53 is markedly increased in human cells in response to expression of the Ad12 E1A proteins and, quite separately, to the Ad12 E1B 54K protein. The behaviour of p53 in these two circumstances has been examined using A549 cells infected with Ad12dl620 (a mutant virus which does not express the larger E1B protein and is replication-defective) and human skin fibroblasts expressing the Ad12 E1B 54K protein (HSF 54K). In normal and E1A-expressing A549 cells, p53 is located predominantly in the nucleus, whereas in the HSF 54K cells it is primarily cytoplasmic as is the Ad12 E1B 54K protein. The half-life of p53 is increased in Ad12dl620-infected A549 cells from about 10 min (in uninfected cells) to 2 hr. The half-life of p53 in HSF 54K cells is even longer—probably in excess of 48 hr. The capacity of p53 to regulate transcription was assessed using a transfected CAT construct linked to p53-responsive elements. p53 transcriptional activity was very low in the HSF 54K cells and in human embryo kidney cells expressing the Ad12 E1B 54K protein (and p53) at high level. It was, however, dramatically increased in response to the p53 expressed as a result of E1A expression. Additionally, MDM2 was present at low level in the HSF 54K cell lines, whilst, as we have previously shown, it is overexpressed in response to infection with Ad12dl620. We conclude that there are two distinct mechanisms for up-regulation of p53 attributable to the adenovirus E1 proteins. When E1A only is present the p53 is nuclear and transcriptionally active and can probably induce apoptosis in the absence of the E1B 19K protein. When the E1B 54K protein is present, however, p53 is transcriptionally inactive and does not induce apoptosis.

The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 9BK protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.

The monoclonal antibody AGF2.3 identifies a nuclear envelope protein that is restricted to certain cell types. In particular, this antigen shows a reduced level of expression during haemopoietic cell maturation. In this study, we have examined the relationship of this protein to known nuclear envelope proteins that have a similar molecular mass. Antigen extraction and immunoelectron microscope studies revealed that the AGF2.3 protein is an integral membrane protein present at both the inner and outer aspects of the nuclear envelope. The protein is not associated with nuclear pores and therefore is distinct from pore complex proteins. The AGF2.3 protein does not have ATPase activity. Therefore, this protein is also distinct from a myosin heavy chain-like ATPase that is associated with the nuclear envelope. The AGF2.3 antibody identifies a novel nuclear envelope protein. Further studies of the biochemical nature of the AGF2.3 protein should provide insight into novel cellular processes at the nuclear envelope relating to the lineage or maturation status of cells.

Adenovirus 12 early region 1A (Ad12 E1A) was expressed inEscherichia coli.Protein was purified in good yield in the presence of 8 M urea and then renatured by dialysis against dilute NH4HCO3buffer. The affinity of this protein for pRb, C-terminal binding protein (CtBP), TATA binding protein (TBP), and SUG1 was similar to, or greater than, that of Ad12 E1A prepared by immunoaffinity chromatography under nondenaturing conditions. While the binding of the 266- and 235-amino-acid (aa) E1A components to TBP showed similar characteristics the larger E1A protein had a higher affinity for CtBP, pRb, and SUG1. Using nuclear magnetic resonance (NMR) spectroscopy it was shown that structural perturbations occurred in the 266-aa protein in the presence of Zn2+consistent with binding—no such changes were seen for the 235-aa protein. Limited proteolysis of the 266- and 235-aa E1A proteins gave rise to comparable polypeptide products, suggesting overall similarities in structure. However, the different affinities of the 266- and 235-aa proteins for the partner proteins and the differences seen in the NMR spectra from the two proteins suggested structural differences.

The human papillomavirus 1 (HPV-1) virion is composed of two virally encoded proteins: a 57,000-molecular-weight polypeptide (57K polypeptide), which is the product of the L1 open reading frame (ORF), and a 78K polypeptide, which is derived from the L2 ORF. The 57K (L1) product, which represents the major structural component, appears to be disulfide cross-linked in virus particles. The 78K (L2) protein is a minor component of the virion and does not appear to be disulfide linked either to the L1 gene product or to itself. Analysis of virus particles banding at different buoyant densities revealed differences in the L2 content of heavy-full and light-full virions. Antiserum prepared against a bacterially expressed fragment of the L1 ORF was found by immunofluorescence to cross-react with HPV-2 and bovine papillomavirus 1 virions in wart sections. No cross-reactivity was observed with antisera prepared against either the N- or C-terminal halves of the L2-encoded protein. Similarly, antisera prepared against purified virus particles (disrupted and nondisrupted) reacted only with an expressed fragment of the L1 ORF and not with either L2-encoded polypeptides or proteins derived from the E1, E2, E4, E6, or E7 ORFs. This indicates that the L1 protein contains the papillomavirus common antigens.

Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose.

It has previously been shown that following viral infection, Ad5 E1A induces cell cycle progression of quiescent rodent cells, leading to DNA synthesis and mitosis. Here we have examined the effect of Ad12 E1A on the cell cycle characteristics of human cells. Human tumor (A549, KB, and HeLa) cells were infected with Ad12dl620, a mutant virus which has a lesion in the E1B gene and essentially expresses only E1A. These infected cells progressed from being largely in G1into S phase, where they arrested. Even up to 96 h postinfection (p.i.) the cells remained blocked in S phase. DNA synthesis did, however, proceed in Ad12dl620-infected cells, giving rise to multiple copies of cellular DNA. Similar results were obtained when primary human skin fibroblasts were infected, although the polyploidy was less marked. The expression of cyclins A, B1, and E in the tumor cells increased appreciably in response to E1A. In contrast, there was a dramatic reduction in the levels of cyclin D1 and D3. Increases in cyclin D1 expression could be detected at very late times p.i. In those cell lines expressing low levels of cdc2 and cdk2 an appreciable increase in expression was seen soon after Ad12 E1A could be detected. The elevated levels of cyclins A, B1, and E were associated with increased protein kinase activity directed against histone H1. An increase in cyclin D1-associated kinase activity against Rb1 was also observed at late times. This deregulation of the cell cycle was not solely dependent on E1A inactivation of Rb, since similar effects were seen in Ad12dl620-infected retinoblastoma (Y-79) cells, implicating p107 and p130 in E1A-mediated changes in cell cycle progression. We propose that the E1A-induced levels of cyclins A, B1, and E by Ad12 E1A in human cells may lead to an uncoupling of S phase from cell cycle progression.