Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts.

The occurrence of DNA double strand breaks induces cell cycle arrest in mortal and immortal human cells. In normal, mortal fibroblasts this block to proliferation is permanent. It depends on the growth regulator p53 and a protein p53 induces, the cyclin dependent kinase inhibitor, p21. We show here that following DNA damage in mortal fibroblasts, the induction of p21 and p53 is to a large degree shortlived. By 8 days after a brief exposure to DNA strand breaking agents, bleomycin or actinomycin D, p53 protein is at baseline levels, while the p53 transactivation level is only slightly above its baseline. By this time the concentration of p21 protein, which goes up as high as 100-fold shortly after treatment, is down to just 2-4-fold over baseline levels. Following the drop in p21 concentration a large increase in the expression level of the tumor suppressor gene p16INK4a is observed. This scenario, where a transient increase in p21 is followed by a delayed induction of p16INK4a, also happens with the permanent arrest that occurs with cellular senescence. In fact, these cells treated with agents that cause DNA double strand breaks share a number of additional markers with senescent cells. Our findings indicate that these cells are very similar to senescent cells and that they have additional factor(s) beside p21 and p53 that maintain cell cycle arrest.

Increased FOXM1 expression can stimulate DNA repair in normal hepatocytes in vivo but also increases nuclear foci associated with senescence.

OBJECTIVES: FOXM1 is a transcription factor that has been shown to promote cell proliferation in many tissues during early development and high cell turnover tissues in adults. In a number of tumour cell lines, enrichment of FOXM1 has been shown to reduce the DNA damage response (DDR) and induction of senescence by a range of DNA-damaging agents, suggesting a role for the protein in DNA repair. Endogenous FOXM1 is expressed at detectable levels in hepatocytes of mice up to 2 weeks of age, but not in older mice. The aim of this investigation has been to better understand the role of the protein in DDR in normal cells in vivo. MATERIALS AND METHODS: Mice with artificially prolonged elevated FOXM1 expression in hepatocytes, were exposed to alkylating diethylnitrosamine. RESULTS: FOXM1-enriched mice had dampened DDR after treatment with this alkylating agent, which was consistent with observed increase in expression of genes involved in DNA repair. Paradoxically, mice with FOXM1 expression, within weeks after exposure to the DNA-damaging agent, had increased levels of potentially senescent hepatocytes with large nuclear foci, containing 53BP1. Similarly, spontaneous accumulation of these cells seen with normal ageing in mice was increased with FOXM1 enrichment. CONCLUSION: Despite its known abilities to promote proliferation and DNA repair, and to reduce ROS, enrichment of FOXM1, as with other oncoproteins, may cause increased persistent DNA lesions and/or senescence in normal murine hepatocytes.

Permanent cell cycle arrest in asynchronously proliferating normal human fibroblasts treated with doxorubicin or etoposide but not camptothecin.

Damage to DNA has been implicated in the induction of permanent cell cycle arrest or premature senescence in normal human fibroblasts. We tested the ability of a group of cancer chemotherapeutic agents or related compounds, which can cause DNA double-strand breaks (DSBs) directly or indirectly, to induce a permanent cell cycle arrest in normal proliferating fibroblasts. A brief treatment with etoposide, doxorubicin, cisplatin, or phleomycin D1 induced a block to S phase entry sustained through 15 days. Lower levels of these drugs did not induce appreciable levels of transient cell cycle arrest. Higher concentrations caused cell death that lacked the DNA degradation characteristic of apoptosis. Camptothecin, an agent that causes DNA single-strand breaks, which are converted to DSBs during S phase, was able to induce an efficient, but only transient, cell cycle arrest in these normal cells. The cells did not enter S phase until after removal of the camptothecin. These findings support the idea that permanent cell cycle arrest and cell death are typical reactions of these normal cells to drugs that can cause DSBs. In addition, we report data consistent with the concept that both actinomycin D and doxorubicin are sequestered by cells and slowly released in active form. This is consistent with the observation that both these drugs bind reversibly to intracellular components.

Site-directed mutant p21 proteins defective in both inhibition of E2F-regulated transcription and disruption of E2F-p130-cyclin-cdk2 complexes.

P21 is a regulatory protein that can contribute to cell cycle arrest by inhibiting the cyclin-dependent-kinases (cdks). However, the mechanism that links the inhibition of the cdk activities and the cell cycle arrest is not well established. To investigate this, we studied a purified endogenous cellular complex which contained E2F (in the form of E2F-4), p130, cyclin, and cdk2. This complex of E2F-p130-cyclin-cdk2 is found mainly in cycling cells and is postulated to be an intermediate that leads to the activation of E2F. We previously showed that p21 could disrupt this complex leading to the accumulation of an E2F-p130 complex and the inhibition of E2F-regulated transcription. We analyzed a group of p21 mutants including those that harbored changes in cyclin- and cdk2-binding motifs. We show that both the cyclin and cdk2 binding motifs of p21 are crucial for the disruption of this endogenous complex of E2F-p130-cyclin-cdk2. This suggests a model where the ability of p21 to inhibit the function of this complex is dependent on interactions with both cyclin and cdk2 molecules. This was substantiated by studies with intact cells. P21 mutants that are impaired in their ability to disrupt the cellular E2F-p130-cyclin-cdk2 complex are also shown to be maximally impaired in the ability to repress E2F-regulated transcription.

The p48 subunit of the damaged-DNA binding protein DDB associates with the CBP/p300 family of histone acetyltransferase.

DDB has been implicated in DNA repair as well as transcription. Mutations in DDB have been correlated with the repair-deficiency disease, xeroderma pigmentosum group E (XP-E). The XP-E cells exhibit deficiencies in global genomic repair, suggesting a role for DDB in that process. DDB also possesses a transcription stimulatory activity. We showed that DDB could function as a transcriptional partner of E2F1. But the mechanism by which DDB stimulates E2F-regulated transcription or carry out its DNA repair function is not understood. To investigate the mechanisms, we looked for nuclear proteins that interact with DDB. Here we show that DDB associates with the CBP/p300 family of proteins, in vivo and in vitro. We suggest that DDB participates in global genomic repair by recruiting CBP/p300 to the damaged-chromatin. It is possible that the histone acetyltransferase activities of the CBP/p300 proteins induce chromatin remodeling at the damaged-sites to allow recruitment of the repair complexes. The observation offers insights into both transcription and repair functions of DDB.

Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression.

All three polyomavirus late mRNAs contain multiple tandem copies of the same nontranslated 57-nucleotide sequence, the late leader, at their 5' ends. We show here that a polyoma variant (ALM) lacking 48 central bases of the 57-base leader unit is nonviable by plaque assay and by a new method for testing virus viability, an immunofluorescence burst assay. ALM is, however, unaffected in early gene expression as measured both by indirect immunofluorescence of large T antigen and by transformation levels of rat F-111 cells. DNA replication in mouse cells is also as wild type, and the defect in ALM is complemented by an early-defective helper virus DNA. ALM does not make detectable levels of late viral proteins and is minimally 200-fold depressed in the accumulation of cytoplasmic polyadenylated late RNA. When the deleted leader sequence of ALM is replaced by a variety of procaryotic sequences, viability almost always returns. Some of the substituted leader variants produce plaques with the same apparent kinetics as wild-type viral DNA. The indication is that the sequence of the polyoma late leader is not important for late gene expression but that it has an essential spacer function on the RNA or DNA level. This spacer function is apparently necessary for late viral RNA transcription, processing, or stability.

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.

The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression.

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.

Misregulated expression of the cyclin dependent kinase 2 protein in human fibroblasts is accompanied by the inability to maintain a G2 arrest following DNA damage.

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.

An adenoviral system for tetracycline-regulated TGF-beta expression mediates a reversible cell cycle arrest.

The ability to regulate the proliferation of normal cells in a reversible manner would be a useful adjunct to some clinical therapies. including many types of cancer chemotherapy and surgery for periodontal regeneration. While the application of recombinant growth factors and cytokines to target cells is a logical approach to regulate cell proliferation, the high turnover rates of these peptide factors often make this approach impractical. Recombinant adenoviral vectors can be used to direct the expression of transgene products such as growth factors in many cell types in vitro and in vivo. We have adapted the tetracycline-regulated expression system to allow regulated transforming growth factor (TGF)-beta1 expression using recombinant adenovirus. We demonstrate that infection with a recombinant TGF-beta-encoding virus system in primary human oral keratinocytes and in a lung epithelial cell line is sufficient to allow a cell cycle arrest that is reversible upon tetracycline addition. This inhibition is efficient even after the infection of a minority of cells in a population. These results highlight the possibility of using low level infection with recombinant adenovirus to cause short-term blocks on cell proliferation.

Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.

Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non-dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22-71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49-71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60-76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49-65 of p21Sdi1 were substituted with amino acids 60-76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.