Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls.

The human hepatitis B virus (HBV) HBx protein is a small transcriptional activator that is essential for virus infection. HBx is thought to be involved in viral hepatocarcinogenesis because it promotes tumorigenesis in transgenic mice. HBx activates the RAS-RAF-mitogen-activated protein (MAP) kinase signaling cascade, through which it activates transcription factors AP-1 and NF-kappa B, and stimulates cell DNA synthesis. We show that HBx stimulates cell cycle progression, shortening the emergence of cells from quiescence (G0) and entry into S phase by at least 12 h, and accelerating transit through checkpoint controls at G0/G1 and G2/M. Compared with serum stimulation, HBx was found to strongly increase the rate and level of activation of the cyclin-dependent kinases CDK2 and CDC2, and their respective active association with cyclins E and A or cyclin B. HBx is also shown to override or greatly reduce serum dependence for cell cycle activation. Both HBx and serum were found to require activation of RAS to stimulate cell cycling, but only HBx could shorten checkpoint intervals. HBx therefore stimulates cell proliferation by activating RAS and a second unknown effector, which may be related to its reported ability to induce prolonged activation of JUN or to interact with cellular p53 protein. These data suggest a molecular mechanism by which HBx likely contributes to viral carcinogenesis. By deregulating checkpoint controls, HBx could participate in the selection of cells that are genetically unstable, some of which would accumulate unrepaired transforming mutations.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

The BCL2 major breakpoint region is a sequence- and cell-cycle-specific binding site of the Ku antigen.

The majority of translocations involving BCL2 are very narrowly targeted to three breakpoint clusters evenly spaced over a 100-bp region of the gene's terminal exon. We have recently shown that the immediate upstream boundary of this major breakpoint region (mbr) is a specific recognition site for single-strand DNA (ssDNA) binding proteins on the sense and antisense strands. The downstream flank of the mbr is a helicase binding site. In this report we demonstrate that the helicase and ssDNA binding proteins show reciprocal changes in binding activity over the cell cycle. The helicase is maximally active in G1 and early S phases; the ssDNA binding proteins are maximally active in late S and G2/M phases. An inhibitor of helicase binding appears in late S and G2/M. Finally, at least one component of the helicase binding complex is the Ku antigen. Thus, a protein with helicase activity implicated in repair of double-strand breaks, variable (diversity) joining recombination, and, potentially, cell-cycle regulation is targeted to the BCL2 mbr.

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

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.