Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.     

Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product

The human retinoblastoma gene (RB1) encodes a protein (Rb) of 105 kilodaltons that can be phosphorylated. Analysis of Rb metabolism has shown that the protein has a half-life of more than 10 hours and is synthesized at all phases of the cell cycle. Newly synthesized Rb is not extensively phosphorylated (it is "underphosphorylated") in cells in the G0 and G1 phases but is phosphorylated at multiple sites at the G1/S boundary and in S phase. HL-60 cells that were induced to terminally differentiate by various chemicals lost their ability to phosphorylate newly synthesized Rb at multiple sites when cell growth was arrested. These findings suggest that underphosphorylated Rb may restrict cell proliferation.     

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.     

Association of human papillomavirus types 16 and 18 E6 proteins with p53

Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.     

Point mutational inactivation of the retinoblastoma antioncogene

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.     

Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.     

马立克氏病病毒p79抗原基因在大肠杆菌中的表达特性分析

马立克氏病病毒（ＭＤＶ）的７９ｋｄ蛋白质是该属３个血清型所共有的抗原．用能表达该蛋白质片段的重组噬菌体Ｌａｍｂｄａｇｔ１１克降ＧＢ－２在宿主菌大肠杆萌Ｙ１０９０菌株中表达该蛋白，免疫转印试验中的确产生了能为对７９ｋｄ蛋白质的单克隆抗体Ｔ８１所识别的表达产物。表达过程中，不论加入或不加蛋白分解酶抑制剂，在ＳＤＳ－ＰＡＧＥ蛋白电泳及Ｗｅｓｔｅｒｎｂｌｏｔ中均未显示分子量大于β－半乳糠甘酶的融合蛋白的存在，而是产生了能为单克隆抗体Ｔ８１识别的分子量分别约为５０ｋｄ，３０ｋｄ，２６ｋｄ的３条多肽，用从凝胶中回收的这３条多肽混合物免疫的小鼠血清，在１：４稀释时，与ＭＤＶ感染的ＤＥＦ细胞在免疫荧光反应中呈阳性反应。此外，用抗β－半乳糖甘酶免疫亲和层析柱杜所得到的提纯蛋白质也不被单克隆抗体Ｔ８１所识别．用和不用ＩＰＴＧ诱导的对照试验表明，在该ＧＢ－２克隆中的ＭＤＶｐ７９基因的表达与Ｌａｍｂｄａｇｔ１１表达外源基因的通常方式不同，不需要ＰＴＧ诱导，而是呈现结构性表达的特征，暗示该蛋白质基因的表达并不使用β－半乳糖苷酶基因启动子。     

Est1p as a cell cycle-regulated activator of telomere-bound telomerase

In Saccharomyces cerevisiae, the telomerase components Est2p, TLC1 RNA, Est1p, and Est3p are thought to form a complex that acts late during chromosome replication (S phase) upon recruitment by Cdc13p, a telomeric DNA binding protein. Consistent with this model, we show that Est1p, Est2p, and Cdc13p are telomere-associated at this time. However, Est2p, but not Est1p, also binds telomeres before late S phase. The cdc13-2 allele has been proposed to be defective in recruitment, yet Est1p and Est2p telomere association persists in cdc13-2 cells. These findings suggest a model in which Est1p binds telomeres late in S phase and interacts with Cdc13p to convert inactive, telomere-bound Est2p to an active form.     

cdc2 gene expression at the G1 to S transition in human T lymphocytes

The product of the cdc2 gene, designated p34cdc2, is a serine-threonine protein kinase that controls entry of eukaryotic cells into mitosis. Freshly isolated human T lymphocytes (G0 phase) were found to have very low amounts of p34cdc2 and cdc2 messenger RNA. Expression of cdc2 increased 18 to 24 hours after exposure of T cells to phytohemagglutinin, coincident with the G1 to S transition. Antisense oligodeoxynucleotides could reduce the increase in cdc2 expression and inhibited DNA synthesis, but had no effect on several early and mid-G1 events, including blastogenesis and expression of interleukin-2 receptors, transferrin receptors, c-myb, and c-myc. Induction of cdc2 required prior induction of c-myb and c-myc. These results suggest that cdc2 induction is part of an orderly sequence of events that occurs at the G1 to S transition in T cells.     

Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.     

Suppression of human colorectal carcinoma cell growth by wild-type p53

Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.     

An antigen produced by splicing of noncontiguous peptides in the reverse order

CD8-positive T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. Here we describe a human minor histocompatibility antigen created by a polymorphism in the SP110 nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order to that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20S proteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation.     

A centrosomal localization signal in cyclin E required for Cdk2-independent S phase entry

Excess cyclin E-Cdk2 accelerates entry into S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. We identified 20 amino acids in cyclin E as a centrosomal localization signal (CLS) essential for both centrosomal targeting and promoting DNA synthesis. Expressed wild-type, but not mutant, CLS peptides localized on the centrosome, prevented endogenous cyclin E and cyclin A from localizing to the centrosome, and inhibited DNA synthesis. Ectopic cyclin E localized to the centrosome and accelerated S phase entry even with mutations that abolish Cdk2 binding, but not with a mutation in the CLS. These results suggest that cyclin E has a modular centrosomal-targeting domain essential for promoting S phase entry in a Cdk2-independent manner.     

DNA ligase: structure, mechanism, and function

DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.     

Rapid destruction of human Cdc25A in response to DNA damage

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.     

Tet-on系统诱导猿猴病毒40T在CHO细胞中的表达

四环素诱导表达系统（Tet-off／Tet-on系统）是比较成熟的真核生物基因诱导表达系统之一,具有高效、无毒、严密开／关功能的特点。猿猴病毒40T（SV40T）是一种病毒癌蛋白,其与肿瘤抑制蛋白p53和Rb结合,并使之失活,从而消除它们抑制细胞生长的功能,使细胞分裂加速,形成肿瘤。利用Tet-on系统首先稳定筛选获得了表达Tet-on系统调节元件rtTA的阳性细胞CHO-pTet-on,再通过稳定筛选又成功得到导入其反应元件的双阳性细胞CHO-pTet-on-pTRE2-SV40T-Hyg,经强力霉素诱导表达了目的基因SV40T,建立了Tet-on基因诱导表达系统的细胞诱导表达研究平台。     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.