The large subunit of replication factor C promotes cell survival after DNA damage in an LxCxE motif- and Rb-dependent manner.

Retinoblastoma (Rb) protein promotes cell survival after DNA damage. We show here that the LxCxE binding site in Rb mediates both cell survival and cell-cycle arrest after DNA damage. Replication factor C (RF-C) complex plays an important role in DNA replication. We describe a novel function of the large subunit of RF-C in promoting cell survival after DNA damage. RF-Cp145 contains an LxCxE motif, and mutation of this motif abolishes the protective effect of RF-Cp145. The inability of wild-type RF-Cp145 to promote cell survival in Rb-null cells is rescued by Rb but not by Rb mutants defective in binding LxCxE proteins. RF-C thus enhances cell survival after DNA damage in an Rb-dependent manner.

The functions of the cdk-cyclin kinase inhibitor p21WAF1.

p21WAF1 plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21WAF1 (hereafter referred to as p21) are the cdk-cyclins which regulate the progression of eukaryotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase delta. p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit DNA synthesis. These distinct properties map to the N-terminal and the C-terminal regions of p21, respectively. Cell cycle arrest in G-1 (G-1 checkpoint) following DNA damage is mediated by p53 and is deficient in p21 null cells. p53 thus upregulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity. Retinoblastoma protein is regulated by cdk-cyclin kinases, and acts as a downstream target of p21 in DNA damage-induced G-1 arrest. Furthermore, accumulating evidence indicates that p21 may play a role in maintaining G-2 arrest after DNA damage. Transcriptional control of p21 by factors other than p53 is critical for growth arrest and for cell differentiation in many instances.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.

The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21(WAF1/Cip1) blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Cellular response to DNA damage. Link between p53 and DNA-PK.

Cells which lack DNA-activated protein kinase (DNA-PK) are very susceptible to ionizing radiation and display an inability to repair double strand DNA breaks. DNA-PK is a member of a protein kinase family that includes ATR and ATM which have strong homology in their carboxy-terminal kinase domain with PL-3 kinase. ATM has been proposed to act upstream of p53 in cellular response to ionizing radiation. DNA-PK may similarly interact with p53 in cellular growth control and in mediation of the response to ionizing radiation.

Jun kinase phosphorylates and regulates the DNA binding activity of an octamer binding protein, T-cell factor beta1.

POU domain proteins have been implicated as key regulators during development and lymphocyte activation. The POU domain protein T-cell factor beta1 (TCFbeta1), which binds octamer and octamer-related sequences, is a potent transactivator. In this study, we showed that TCFbeta1 is phosphorylated following activation via the T-cell receptor or by stress-induced signals. Phosphorylation of TCFbeta1 occurred predominantly at serine and threonine residues. Signals which upregulate Jun kinase (JNK)/stress-activated protein kinase activity also lead to association of JNK with TCFbeta1. JNK associates with the activation domain of TCFbeta1 and phosphorylates its DNA binding domain. The phosphorylation of recombinant TCFbeta1 by recombinant JNK enhances the ability of TCFbeta1 to bind to a consensus octamer motif. Consistent with this conclusion, TCFbeta1 upregulates reporter gene transcription in an activation- and JNK-dependent manner. In addition, inhibition of JNK activity by catalytically inactive MEKK (in which methionine was substituted for the lysine at position 432) also inhibits the ability of TCFbeta1 to drive inducible transcription from the interleukin-2 promoter. These results suggest that stress-induced signals and T-cell activation induce JNK, which then acts on multiple cis sequences by modulating distinct transactivators like c-Jun and TCFbeta1. This demonstrates a coupling between the JNK activation pathway and POU domain proteins and implicates TCFbeta1 as a physiological target in the JNK signal transduction pathway leading to coordinated biological responses.

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis.

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.

Cloning of a T-cell receptor beta-chain enhancer binding protein.

Several eukaryotic DNA binding proteins have been isolated by screening lambda expression libraries with DNA probes containing their binding site. This strategy has been employed to isolate clones of the factor that interacts with the T-cell receptor beta-chain enhancer motif. A cDNA clone encoding a protein similar to YB-1 has been isolated with this. It seems probable that this protein YB, might interact with other proteins and regulate the transcription of the T-cell receptor beta-chain gene.

Role for cyclin A-dependent kinase in DNA replication in human S phase cell extracts.

Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21(Waf1) mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.

p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity.

Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21Cip1/Waf1/Sdi1 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase delta. The kinase inhibitory domain maps to the N-terminus (1-82) and contains the cdk2 binding site (28-82). We have generated a panel of deletion mutants of p21. A functional characterization of p21 mutants in the N-terminal domain reveals that cyclins bind to this domain independently of cdk2. Correlating with these results we find that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.

Apoptosis and the cell cycle.

Apoptosis is a genetically controlled response by which eukaryotic cells undergo programmed cell death. This phenomenon plays a major role in developmental pathways (1), provides a homeostatic balance of cell populations, and is deregulated in many diseases including cancer. Control of cell number is determined by an intricate balance of cell death and cell proliferation. Accumulation of cells through suppression of death can contribute to cancer and to persistent viral infections, while excessive death can result in impaired development and in degenerative diseases. Identification of genes that control cell death, and understanding of the impact of apoptosis in both development and disease has advanced our knowledge of apoptosis in the past few years. There appears to be a linkage between apoptosis and cell cycle control mechanisms. Elucidating the mechanisms that link cell cycle control with apoptosis will be of key importance in understanding tumour progression and designing new models of effective tumour therapy.

Immunoregulatory activity of a T-cell receptor alpha chain demonstrated by in vitro transcription and translation.

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor alpha (TCR alpha) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCR alpha chain (A1.1 TCR alpha) suspected of having such activity. We found that TCR alpha, but not TCR beta, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCR alpha cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the alpha chain (VJ alpha), at least the first 25 amino acids of the alpha chain of the constant (C) region (C alpha) were present. Addition of an irrelevant protein sequence to the VJ alpha failed to impart activity to the molecule, suggesting that the C alpha requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCR alpha molecules, and the possible physiological relevance of these observations is considered.

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Cell cycle control of DNA replication.

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (cdk), which phosphorylate and activate proteins that execute events critical to cell cycle progression. In mammalian cells cdk2-cyclin A has a role in S phase. Many replication proteins are potential substrates for this cdk kinase, suggesting that initiation, elongation and checkpoint control of replication could all be regulated by cdk2. The association of PCNA, a replication protein, with cdk-cyclins during G-1 to S phase transition and with cdk-cyclin inhibitors, adds an interesting complexity to regulation of DNA replication.

A novel POU domain protein which binds to the T-cell receptor beta enhancer.

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.

p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene.

The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients. In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene. This enhancer is essential for expression of the TCR beta gene. The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity. The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity. The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe. The gene encodes a protein which binds the E3 motif in a sequence-specific manner. The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer. Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants. Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression.

Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.