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Objective: To elucidate the biological function of BAP18 (BPTF-associated protein of 18 kDa) in non-small-cell lung carcinoma (NSCLC) and the molecular mechanism., Patients and Methods: Relative levels of BAP18 in NSCLC tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and its influence on pathological characteristics of NSCLC patients was analyzed. Correlation between BAP18 and Ki67 levels in NSCLC was assessed by Pearson correlation test. Furthermore, Kaplan-Meier curves were depicted for revealing survival difference in NSCLC patients expressing high or low level of BAP18. Relative levels of BAP18, CCND1, CCND2 and CCND3 in A549 and H1299 cells transfected with siBAP18 were determined, as well as colony number. In addition, after knockdown of protein level of BAP18 in A549 and H1299 cells by lentivirus transfection, cell cycle progression was examined. Co-regulation of BAP18 and CCND1/2 on cell growth of NSCLC was finally detected., Results: BAP18 was upregulated in NSCLC tissues, especially cases with advanced stage (III-IV) or large tumor size (>5 cm). BAP18 was closely linked to tumor size, TNM staging and lymphatic metastasis in NSCLC. Knockdown of BAP18 reduced transcriptional levels of CCND1 and CCND2 in A549 and H1299 cells. Furthermore, knockdown of BAP18 delayed transition from G1 to S phase, and weakened growth of NSCLC cells., Conclusions: BAP18 triggers the progression of NSCLC by regulating transcriptional activities of CCND1/2, which may be a potential target for the treatment and diagnosis of NSCLC.

DNA repair is critical for the maintenance of genome stability. Upon genotoxic stress, dysregulated DNA repair may induce apoptosis. Translin-associated factor X (TRAX), which was initially identified as a binding partner of Translin, has been implicated in genome stability. However, the exact role of TRAX in DNA repair remains largely unknown. Here, we showed that TRAX participates in the ATM/H2AX-mediated DNA repair machinery by interacting with ATM and stabilizing the MRN complex at double-strand breaks. The exogenous expression of wild-type (WT) TRAX, but not a TRAX variant lacking the nuclear localization signal (NLS), rescued the vulnerability of TRAX-null mouse embryo fibroblasts (MEFs). This finding confirms the importance of the nuclear localization of TRAX in the repair of DNA damage. Compared with WT MEFs, TRAX-null MEFs exhibited impaired DNA repair (for example, reduced phosphorylation of ATM and H2AX) after treatment with ultra violet-C or γ-ray irradiation and a higher incidence of p53-mediated apoptosis. Our findings demonstrate that TRAX is required for MRN complex-ATM-H2AX signaling, which optimizes DNA repair by interacting with the activated ATM and protects cells from genotoxic stress-induced apoptosis.

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) expression is often silenced in many types of human tumors. However, the relationship between RIZ1 expression and malignant meningiomas remains unclear. Here we have found for the first time that the expression of RIZ1 genes are associated with meningiomas progression through extensive analyses of Affymetrix GeneChip microarray data. Further validation methods for gene expression included quantitative PCR (qPCR), western blot and immunohistochemistry analysis, and these methods confirmed that RIZ1 is significantly downregulated in malignant meningioma tissues, as compared with benign meningiomas. In addition, malignant meningioma cells were stably transfected with ectogenic RIZ1 using Lentivirus-mediated transfection, and the transfections were followed by an in vitro 5-bromo-2-deoxyuridin incorporation assay, colony formation assay, cell cycle analysis, invasive analysis, apoptotic assay and western blot analysis. Our results demonstrate that the forced expression of RIZ1 in a malignant meningioma cell line inhibited cellular proliferation and arrested the cells in the G2/M phase of the cell cycle. We also confirmed that overexpression of RIZ1 may induce apoptosis of malignant meningioma cells. Furthermore, RIZ1 overexpression in malignant meningioma cells was associated with the downregulation of c-myc expression. These results from our study indicate that RIZ1 expression is significantly downregulated as the formation of meningiomas progressed, and suggest that RIZ1 may represent a promising candidate tumor suppressor gene that contributes to malignant meningiomas.

Background: Hereditary non-polyposis colorectal cancer (HNPCC) is characterized genetically by germline mutations in DNA mismatch repair (MMR) genes. Immunohistochemistry (IHC) has high sensitivity and specificity for identifying MMR-deficient tumours. This study investigated the clinical presentations and frequency of HNPCC in Taiwan by combined Amsterdam II criteria (AC-II) and IHC., Methods: In 1995-2003, 7108 patients with primary colorectal cancer registered in Chang Gung Memorial Hospital's Colorectal Cancer Registry were screened using AC-II. Tumour specimens were analysed for MMR protein expression by IHC, and relevant clinicopathological details were documented., Results: Some 83 patients fulfilled the AC-II. Clinicopathologically, 43 patients (52 per cent) had proximal tumours, ten (12 per cent) had poorly differentiated cancers, 17 (20 per cent) had mucinous adenocarcinoma and 51 (61 per cent) had stage I-II tumours. Seventeen patients developed second primary colonic and extracolonic cancers over a mean 7.2-year follow-up. Immunohistochemically, 58 patients were MMR protein deficient. They had a significantly earlier age of onset (P < 0.001), more proximal tumour location (P = 0.002), less advanced tumour stage (P = 0.008) and more second primary cancers (P = 0.017) compared with MMR-competent patients., Conclusion: These data show significant differences in clinical features between MMR protein-deficient and MMR competent subgroups., (Copyright (c) 2007 British Journal of Surgery Society Ltd.)

Ionizing radiation (IR) induces DNA breakage to activate cell cycle checkpoints, DNA repair, premature senescence or cell death. A master regulator of cellular responses to IR is the ATM kinase, which phosphorylates a number of downstream effectors, including p53, to inhibit cell cycle progression or to induce apoptosis. ATM phosphorylates p53 directly at Ser15 (Ser18 of mouse p53) and indirectly through other kinases. In this study, we examined the role of ATM and p53 Ser18 phosphorylation in IR-induced retinal apoptosis of neonatal mice. Whole-body irradiation with 2 Gy IR induces apoptosis of postmitotic and proliferating cells in the neonatal retinas. This apoptotic response requires ATM, exhibits p53-haploid insufficiency and is defective in mice with the p53S18A allele. At a higher dose of 14 Gy, retinal apoptosis still requires ATM and p53 but can proceed without Ser18 phosphorylation. These results suggest that ATM activates the apoptotic function of p53 in vivo through alternative pathways depending on IR dose., (2004)

The choice between growth arrest and apoptosis is made during differentiation, leading to survival with permanent arrest (e.g. neurons), or to death (e.g. epithelium). Genotoxic stress can also cause growth arrest or apoptosis, in addition to the activation of cell cycle checkpoint pathways. The p53 tumour suppressor can simulate growth arrest and apoptosis in response to DNA damage. Thus, p53 alone is not sufficient to specify these two mutually exclusive fates in damaged cells. The retinoblastoma tumour suppressor protein (RB) is a necessary downstream effector in p53-mediated growth arrest. RB inhibits E2F and the nuclear c-Abl tyrosine kinase. Interestingly, E2F activates the transcription of p73 mRNA and c-Abl stabilizes the p73 protein and activates its pro-apoptotic function. Because of RB, the c-Abl/p73 apoptosis pathway is activated in S/G(2) cells but not in G(1) cells. Taken together, the current data suggests RB to be an important player in directing the choice between permanent arrest and apoptosis. The antagonism between RB and c-Abl/p73 may modulate the function of p53 to direct the choice between growth arrest and apoptosis in DNA damaged cells.

E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have onco-suppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.

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.

Adeno-associated virus (AAV) inhibits the induction of host DNA synthesis by simian virus 40 (SV40) large-tumour (T) antigen, mediated through AAV-encoded 'Rep' regulatory proteins. Rep proteins are normally synthesized by AAV-infected cells only in the presence of adenovirus. However, we observed a low level of Rep protein expression in SV40 transformed cells even in the absence of helper virus. In an effort to understand the functional interaction between SV40 T antigen and regulators of AAV rep expression, we evaluated Rep protein production by cell lines transformed with various T antigen mutants known to vary in their induction of host DNA synthesis. We observed Rep protein expression proportional to SV40-induced host DNA synthesis, as measured previously for these T antigen mutants in the absence of AAV, suggesting that rep gene expression - although it opposes the oncogenic stimulation of cell cycling by SV40 - may itself be elicited by host DNA synthesis. To test this, we employed two inhibitors of DNA synthesis: hydroxyurea, which acts by depleting deoxyribose nucleotide triphosphate pools, and aphidicolin, a specific inhibitor of DNA polymerases alpha and delta. Each inhibitor markedly and significantly reduced Rep protein levels, both in immortal cells transformed by wild-type T antigen and in normal human fibroblasts, confirming the dependence of Rep protein expression on host DNA synthesis., (Copyright 2001 S. Karger AG, Basel)

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.

The crystal structure of the A-B domain of RB has defined the binding pocket for the LXCXE peptide motif. Using the crystal structure as a guide, we have inactivated the LXCXE-binding pocket by replacing N757 with Phe [to obtain RB(N757F)]. RB(N757F) does not bind to viral oncoproteins but retains the ability to bind and inhibit E2F. RB(N757F) is less effective than the wild-type RB [RB(WT)] in repressing E2F-regulated transcription, and its repression activity is not affected by trichostatin A, an inhibitor of histone deacetylases. However, RB(N757F) is as effective as RB(WT) in suppressing cell growth. Interestingly, RB(N757F) cannot establish an irreversible growth arrest in differentiated myocytes. Differentiated myocytes with RB(WT) become refractory to serum. By contrast, differentiated myocytes with RB(N757F) undergo DNA synthesis and phosphorylate RB(N757F) in response to serum, despite a high level of p21Cip1 expression. Mutation of the phosphorylation sites in RB(N757F) rescued its defect and allowed myocytes to permanently withdraw from the cell cycle. These results demonstrate that it is possible to inactivate the LXCXE-binding pocket without compromising the overall integrity of RB. Moreover, the LXCXE-binding pocket is dispensable for the intrinsic growth suppression function of RB. However, the LXCXE-binding function is essential for RB to establish the serum-refractory state in differentiated myocytes.

The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun-N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38alpha(-/-) embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes.

The p53 gene is the most frequently mutated gene in human cancer. The identification of two homologues, p63 and p73, revealed that p53 is a member of a family of related transcription factors. Given that they share amino acid sequence identity reaching 63% in the DNA-binding domain, p53, p63 and p73 should have redundant functions in the regulation of gene expression. Indeed, p73 can activate p53-regulated genes and suppress growth or induce apoptosis. Moreover, p53 and p73 are both induced by DNA damage - albeit through distinct mechanisms. Other evidence, however, suggests that p63 and p73 are important for regulation of normal development. An extended C-terminal region, not found in p53, is alternatively spliced in p63 and p73. Within this C-terminal extension is a sterile &agr; motif (SAM) previously found in other proteins that regulate development. The p63-deficient mice showed developmental abnormalities. Interestingly, the human p63 gene is mutated in children who have the disease Ectrodactyly, Ectodermal dysplasia and facial Clefts (EEC) syndrome, and the disease phenotype is similar to the one of p63-deficient mice. The p63 and p73 genes are rarely mutated in human cancer, although p73 loss is observed in neuroblastoma and a subtype of T-cell lymphoma. p53, p63 and p73 appear to have overlapping and distinct functions: p53 regulates the stress response to suppress tumors; p63 is essential for ectoderm development; and p73 might regulate both the stress response and development. Because p53 and p73 are linked to different upstream pathways, this family of transcription factors might regulate a common set of genes in response to different extracellular signals and developmental cues.

LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, we have determined the subcellular distribution of LIMK1 protein within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 was concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity was detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals were not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincided with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurred long after these synapses first formed. In the adult ventral spinal cord, LIMK1 was concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulated at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses., (Copyright 2000 Wiley-Liss, Inc.)

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers.1 p53 is a sequence-specific transcription factor and plays a critical role in activating the expression of genes involved in cell cycle arrest or apoptosis under conditions of genotoxic stress.2,3 For over two decades, p53 was thought to be the only gene of its kind in the vertebrate genomes. This strong conviction, which was widely accepted in the p53 field, has now been proven to be incorrect. Two genes, referred to as p63 and p73, have been found to encode proteins that share a significant amino-acid identity in the transactivation domain, the DNA binding domain, and the oligomerization domain with p53. In the short period since their cloning, a number of investigators have reported on the structure, the function and the regulation of p63 and p73. This review summarizes the current information on the p63 and the p73 genes, with a focus on the differences between the three members in this newly defined p53-gene family.

Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA virus belonging to the parvoviridae family. Onco-suppressive properties of AAV against adenovirus, a DNA tumor virus, have been well documented. Rep78, a major regulatory protein of AAV, is believed to be responsible for its antioncogenic properties. Most DNA tumor viruses disturb the cell cycle pathways by essentially abrogating the functions of p53. Here we present evidence that AAV acts as an antiproliferative agent against adenovirus by protecting the adenoviral-mediated degradation of p53 as confirmed by both Western blot analysis and immunoprecipitation analysis with anti-p53 antibody. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. Furthermore, the binding of purified p53 to the AAV Rep78 affinity column confirms their interaction. These results document for the first time that the antiproliferative effects of AAV against adenovirus are mediated, at least in part, by the interaction of AAV Rep78 with p53.

Cancer chemotherapeutic agents such as cisplatin exert their cytotoxic effect by inducing DNA damage and activating programmed cell death (apoptosis). The tumour-suppressor protein p53 is an important activator of apoptosis. Although p53-deficient cancer cells are less responsive to chemotherapy, their resistance is not complete, which suggests that other apoptotic pathways may exist. A p53-related gene, p73, which encodes several proteins as a result of alternative splicing, can also induce apoptosis. Here we show that the amount of p73 protein in the cell is increased by cisplatin. This induction of p73 is not seen in cells unable to carry out mismatch repair and in which the nuclear enzyme c-Abl tyrosine kinase is not activated by cisplatin. The half-life of p73 is prolonged by cisplatin and by co-expression with c-Abl tyrosine kinase; the apoptosis-inducing function of p73 is also enhanced by the c-Abl kinase. Mouse embryo fibroblasts deficient in mismatch repair or in c-Abl do not upregulate p73 and are more resistant to killing by cisplatin. Our results indicate that c-Abl and p73 are components of a mismatch-repair-dependent apoptosis pathway which contributes to cisplatin-induced cytotoxicity.

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.

Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G1/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression.

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (i) the A/B pocket, which binds proteins with the LXCXE motif; (ii) the C pocket, which binds the c-Abl tyrosine kinase; and (iii) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. We have previously shown that LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). In this report, we show that the E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB.

A mathematical model integrating the roles of cyclin D, cdk4, cyclin E, cdk2, E2F and RB in control of the G1 phase of the cell cycle is described. Experimental results described with murine embryo fibroblasts (MEFs), either Rb+/+ or Rb-/-, and with the RB-deficient osteosarcoma cell line, Saos-2, served as the basis for the formulation of this mathematical model. A model employing the known interactions of these six proteins does not reproduce the experimental observations described in the MEFs. The appropriate modelling of G1 requires the inclusion of a sensing mechanism which adjusts the activity of cyclin E/cdk2 in response to both RB concentration and growth factors. Incorporation of this sensing mechanism into the model allows it to reproduce most of the experimental results observed in Saos-2 cells, Rb-/- MEFS, and Rb+/+ MEFs. The model also makes specific predictions which have not been tested experimentally.

Puzzling new information indicates an inadequacy in our understanding of the retinoblastoma protein (RB). RB and the transcription factor E2F appear to be collaborators. RB-E2F interaction is necessary but not sufficient for growth suppression. Unbecoming of a tumor suppressor, RB has an active role in antagonizing the death response. How RB integrates its multiple functions into a tumor suppression program is still an open issue.

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.

The retinoblastoma tumor suppressor protein, RB, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RB from its targets is observed as RB becomes phosphorylated during G1/S progression. There are 16 Cdk consensus phosphorylation sites in RB. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RB. Using RB mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RB. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RB are each regulated by distinct Cdk phosphorylation sites.

The growth suppression function of the retinoblastoma protein (RB) is mediated by its interaction with a variety of cellular proteins. RB contains at least two protein-binding pockets: the large A/B pocket, which interacts with E2F and the D-type cyclins, and the C pocket, which interacts with the nuclear c-Abl tyrosine kinase. The large A/B pocket and the C pocket are shown here to be functionally distinct and can be occupied simultaneously. A complex containing E2F, RB, and c-Abl is detected in vivo and can be assembled in vitro. We propose that the biological activity of RB not only depends on the inhibition of its targets but also on its ability to properly assemble specific protein complexes. Consistent with this hypothesis, a fragment of RB, SE delta, containing only the C pocket is shown to act as a dominant-negative inhibitor of RB function. SE delta does not have growth inhibitory activity of its own. When coexpressed with full-length RB, SE delta does not disrupt the RB-E2F or RB-D2 complexes nor does it affect the expression, phosphorylation, or nuclear tethering of the full-length RB. SE delta does compete with RB for binding to c-Abl and is fully capable of inhibiting the c-Abl tyrosine kinase. Thus, SE delta can inactivate RB while maintaining the inhibition of E2F and c-Abl. These results suggest that the inhibition of RB-binding proteins is not sufficient to suppress cell growth and that the assembly of RB-mediated protein complexes is also important for the promotion of cell-cycle arrest.

We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's sarcomas and peripheral primitive neuroectodermal tumors and cells lines. Type 1 and 2 EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples. Five patients with cytogenetically characterized rearrangements of chromosome 22 that did not involve chromosome 11 were included in these studies. A novel EWS-Fli1 in-frame isoform fusing EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12) translocation. Three in-frame isoforms of a novel hybrid transcript derived from the fusion of EWS with the ETS domain of the human erg gene were identified in patient samples and a cell line with cytogenetically unidentified or cryptic translocations involving chromosomes 21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene on chromosome 21 in a patient sample with monosomy 22. Our results provide new information about the involvement of EWS in small round cell tumors involving exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETS family of transcription factors. These studies emphasize the utility of reverse transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic tools for differential diagnosis among small round cell tumors.

The highly conserved octamer sequence ATGCAAAT or its inverse complement found in all human and murine immunoglobulin gene promoters has been demonstrated to be necessary in the lymphoid-specific transcription by deletion analysis. Trans-acting factors that interact with the octamer motif are thought to be involved in this tissue-specific expression. Using a gel mobility shift assay, we have identified both lymphoid-specific and ubiquitous nuclear factors that interact with a human gamma 1 heavy chain gene promoter region containing the octamer motif, consistent with the results obtained with murine heavy or light chain promoter regions. We have purified an octamer binding protein from human B cells by sequence-specific DNA affinity chromatography. Renaturation of gel-purified protein allowed the identification of a polypeptide with a molecular weight of 74 kilodaltons (kD) that is capable of recognizing and binding to the octamer motif. This 74 kD protein seems to be also present in T-cells and non-lymphoid cells. The possible function of the factor is discussed.