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Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here, we show that Xenopus M18BP1 localizes to centromeres during metaphase-prior to CENP-A assembly-by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase., (© 2019 The Authors.)

Centromeric chromatin defines the site of kinetochore formation and ensures faithful chromosome segregation. Centromeric identity is epigenetically specified by the incorporation of CENP-A nucleosomes. DNA replication presents a challenge for inheritance of centromeric identity because nucleosomes are removed to allow for replication fork progression. Despite this challenge, CENP-A nucleosomes are stably retained through S phase. We used BioID to identify proteins transiently associated with CENP-A during DNA replication. We found that during S phase, HJURP transiently associates with centromeres and binds to pre-existing CENP-A, suggesting a distinct role for HJURP in CENP-A retention. We demonstrate that HJURP is required for centromeric nucleosome inheritance during S phase. HJURP co-purifies with the MCM2-7 helicase complex and, together with the MCM2 subunit, binds CENP-A simultaneously. Therefore, pre-existing CENP-A nucleosomes require an S phase function of the HJURP chaperone and interaction with MCM2 to ensure faithful inheritance of centromere identity through DNA replication., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell-cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Faithful transmission of genetic information during cell division requires attachment of chromosomes to the mitotic spindle via the kinetochore. In vitro reconstitution studies are beginning to uncover how the kinetochore is assembled upon the underlying centromere, how the kinetochore couples chromosome movement to microtubule dynamics, and how cells ensure the site of kinetochore assembly is maintained from one generation to the next. Here we give special emphasis to advances made in Xenopus egg extract, which provides a unique, biochemically tractable in vitro system that affords the complexity of cytoplasm and nucleoplasm to permit reconstitution of the dynamic, cell cycle-regulated functions of the centromere and kinetochore.

Faithful genome segregation depends on the functions of the eukaryotic centromere, which is characterized by the histone variant CENP-A. Gene replacement in human cells and fission yeast has now been used to show how CENP-A biochemically encodes centromere identity, as well as reveal an unexpected role for CENP-B in centromere function.

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.

Maturation of eukaryotic tRNA molecules requires nuclear processing as well as nuclear and cytoplasmic modification of specific nucleotides. Nucleotide modifications within the anticodon are found in the majority of all tRNAs and are among the last maturation events to occur in vivo. We show that 5' and 3' processing of SP6 polymerase-generated transcripts are not necessary for the in vitro modification of A----I in the anticodon of tRNA(Ala).

Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture. Methyl methanesulfonate (MMS) was used to convert premalignant squamous cell carcinoma (SCC) cell line SCC-83-01-82 to a malignant phenotype. The MMS-treated SCC-83-01-82 cells (MMS-SCC-83-01-82) produced progressively growing tumors in 5 of 11 splenectomized BALB/c nude mice within 3-5 months. A cell line, designated SCC-83-01-82 CA, was established in vitro from one of the mouse tumors and was repassaged successively. This SCC-83-01-82 CA cell line was aggressively tumorigenic. A tumor greater than or equal to 2.0 cm in size was present within a month, as opposed to the 3-5 months required for the tumors produced by the MMS-SCC-83-01-82 cells. Examination of frozen cross sections by in situ hybridization revealed that focal areas of the tumor produced by the MMS-SCC-83-01-82 cells expressed MYC and HRAS mRNA. However, by the third passage in vivo, the levels of expression of the corresponding genes in the mouse tumors were undetectable. Blot-hybridization analysis of the RNA from the MMS-SCC-83-01-82 cells and the subsequently derived tumors and cells did not indicate any consistent overexpression of MYC, HRAS, or KRAS. Restriction fragment length polymorphism analysis of both MYC and HRAS genes revealed neither rearrangement nor amplification of MYC nor point mutation in the 11th or 12th codon of HRAS. The data suggest that alterations in MYC and HRAS were not directly involved in either the initial transformation or MMS-induced tumorigenic conversion of the SCC-83-01-82 cell line. Persistence of tumorigenicity after reisolation of the MMS-converted premalignant SCC-83-01-82 cells did not disappear immediately following the treatment with MMS.

A variety of compounds inhibit the growth and induce differentiation of human promyelocytic leukemia (HL-60) cells. HL-60 subclones that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can also be induced to differentiate with purine analogs. Mechanisms by which purine analogs induce differentiation offer unique possibilities for cancer chemotherapy. We have studied the effect of the purine analog 6-ethylmercaptopurine (e6MP) on the growth and induction of differentiation in both wild-type and HGPRT-deficient HL-60 cells. We have previously shown that e6MP inhibits cell growth in both wild-type and HGPRT-deficient HL-60 cells without activation through salvage pathways. In this report we evaluate the effect of e6MP on c-myc mRNA expression. c-Myc mRNA, which is amplified in HL-60 cells, has been shown to play a role in the induction of granulocytic differentiation in HL-60 cells. e6MP transiently down-regulates c-myc mRNA in wild-type cells but has no effect on c-myc mRNA expression in HGPRT-deficient HL-60 cells. Despite the differential effects of e6MP on c-myc mRNA, both wild-type and HGPRT-deficient HL-60 cells appear to engage in terminal differentiation. The morphological changes and nonspecific esterase activity induced by e6MP suggest differentiation down the monocytic pathway. However, early monocytic markers such as the rapid induction of c-fos and the stabilization of c-fms mRNA are not observed. In addition, e6MP inhibits TPA-induced monocytic/macrophage differentiation as characterized by stabilization of c-fms mRNA and cellular adherence.

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen-positive cells was not uniform throughout the tumor. Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC-83-01-82, isolated by passage through soft agar, was treated with 50 micrograms/ml of methyl methane sulfonate (MMS). These MMS-converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c-myc and H-ras mRNA expression. Serial passage of the MMS-converted tumors in vivo was accompanied by consistent enhanced c-myc expression. However, the levels of H-ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c-myc and H-ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC-83-01-82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c-myc and ras gene expression.

Antibodies derived from scleroderma patients positive for the extractable 70-kDa antigen were shown to react with topoisomerase I. Topoisomerase I was identified by molecular size and by antibody inhibition of the topoisomerase I-specific relaxation of supercoiled plasmid DNA. By using in situ localization by indirect fluorescence, we found topoisomerase I preferentially located in the centromeric regions of the mouse G2-phase cells and chromosomes, while the distribution in human cells is much more dispersed. Moreover, comparison of a published consensus sequence for topoisomerase I binding with mouse satellite DNA revealed a high degree of homology. The localization of topoisomerase I in the centromeres of mouse cells in the later part of the cell cycle and prior to anaphase suggests functional involvement in mitosis.

Chromatin may be attached to the nuclear envelope through interaction of the nuclear membrane lamins A, B, and C. Such a hypothesis requires that these proteins are present in all cells with chromatin attachment to the nuclear envelope. We have investigated the distribution of the lamins during spermatogenesis in mouse, which exhibits extremes in nuclear envelope structural changes. By immunohistochemical techniques using human auto-antibodies and monoclonal antibodies against these molecules, we found that the lamins persist through all stages of spermatogenesis, though in highly variable amounts. They are also present during meiotic prophase (pachytene) when chromosomes are only locally attached to the nuclear envelope, analogous to the early prophase of somatic cells. Restructuring of the early spermatid nuclear envelope is accompanied by the appearance of a new lamin at the acrosomal fossa. In the epididymal spermatozoon the distribution of different lamins varies markedly over the nucleus suggesting special structural functions. The presence of lamins throughout spermatogenesis supports the concept that they are a general feature of the nuclear envelope structure, even where a lamina is not recognizable ultrastructurally.

The similarity of the Arrhenius plots relating temperature to messenger RNA (mRNA) transport from intact and membrane-denuded rat liver nuclei demonstrates that the ATP and cytosol-dependent transport is independent of the lipid phase of the nuclear membrane. This temperature dependence of RNA release was confirmed for alpha 2u-globulin mRNA by use of a recombinant DNA probe. Ribosomal RNA (rRNA) release showed a similar temperature dependence, suggesting that both mRNA and rRNA share a common temperature-sensitive step. The kinetics of RNA release at different temperatures suggest that RNA transport from mammalian cell nuclei is a rate-controlled rather than a graded unlocking phenomenon. The processing of mRNA precursors also exhibits a temperature dependence as shown by the linear increase in the ratio of total alpha 2u-globulin RNA to alpha 2u-globulin precursor as a function of time at 30 degrees C but not at 14 degrees C in spite of residual transport at the lower temperature. This temperature dependence of mRNA processing was confirmed by Northern blot analysis of the nuclear RNA following a 45 min incubation. Thus, both the processing and transport of RNA show temperature-sensitive steps when analyzed in cell-free systems derived from mammalian cells.

A chromogenic method using biotinylated secondary antibodies and peroxidase-coupled avidin for screening cDNA expression libraries is described. This method offers increased sensitivity over peroxidase-coupled secondary antibodies and rapid processing of samples, and avoids preparation and handling of radioactive materials. All materials for the chromogenic assay are available commercially and the method offers a fast and easy way to screen lambda and plasmid expression libraries with mono- and polyclonal antibodies.

A cDNA clone for mouse pro alpha 1(I) collagen has been isolated and sequenced. A 1.8-kb PstI fragment spans nucleotides -1991 to -159 of the alpha-domain of mouse pro alpha 1(I) collagen.

A variety of purine analogs inhibit the growth and induce the differentiation of human promyelocytic leukemia (HL-60) cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mechanisms by which purine analogs induce differentiation offer unique potential for cancer chemotherapy. The guanine analogs, 6-thioguanine and 8-azaguanine, induce granulocytic differentiation of HGPRT-deficient HL-60 promyelocytes. Although these compounds are useful as model purine analogs that induce differentiation in HGPRT-deficient HL-60 cells, they suffer the disadvantage that they are highly cytotoxic to wild-type cells. We studied the effect of the hypoxanthine analog 6-ethylmercaptopurine on wild-type and HGPRT-deficient HL-60 cells. 6-Ethylmercaptopurine inhibits growth and produces a specific terminal end-cell in both types of HL-60 cells. The mechanism appears to be independent of the normal modes of cytotoxic activation through HGPRT or adenine phosphoribosyltransferase (APRT), since no new peaks were seen in HPLC chromatograms of the nucleotide pools. Furthermore, hypoxanthine and adenine failed to prevent growth inhibition by 6-ethylmercaptopurine, and inhibition of IMP dehydrogenase and the consequential alteration of the guanine nucleotide pools does not appear to be involved. The mechanism differs from that of guanine analog-induced differentiation in HGPRT-deficient HL-60 cells.

The ability of a reconstituted cell-free system to transport mRNA as a ribonucleoprotein particle has been examined. Poly(A) messenger ribonucleoproteins (mRNPs), UV cross-linked after release from isolated liver nuclei in a cell-free system, exhibited a buoyant density of 1.33 g/cm3 in cesium sulfate and 1.47 g/cm3 in cesium chloride, values identical to those of poly(A) mRNP isolated directly from liver polysomes. Furthermore, the in vivo and in vitro transported mRNP showed a similar degree of resistance to RNase digestion and had sedimentation coefficients approximately 2.5 times that of the isolated mRNA. Release of both total mRNA and alpha 2 mu-globulin mRNA was proportional to the concentration of a specific cytoplasmic protein. Removal of the transport proteins from the cytosol with streptomycin sulfate provided a basal system incapable of supporting the active transport of alpha 2 mu-globulin mRNA. Hybridization of released RNA with a recombinant probe specific for intron 6 of alpha 2 mu-globulin showed that intron sequences were retained within the nucleus under optimal alpha 2 mu-globulin mRNA transport conditions and that the transported alpha 2 mu-globulin mRNA was of mature size.

rRNA release from isolated liver nuclei has been analyzed in a reconstituted cell-free system using density-gradient analysis and hybridization to a specific recombinant DNA probe to monitor the process. The cell-free system was shown previously to be energy- and cytosol-dependent and to support the formation and release of functional ribosomal subunits. The release of rRNA is now shown to have an absolute dependence on a 70000 dalton cytosol protein. Although in vivo studies suggest that chronic administration of thioacetamide may block formation of a protein involved in the nucleocytoplasmic transfer of ribosomes, the 70000 dalton transport factor is not affected by the treatment. Rather the defect appears to be localized to the nucleus, since it cannot be reversed with normal cytosol from a homologous source. Early stages of nRNA processing appear to be affected by thioacetamide, although additional effects on transport are not ruled out.

Treatment of rats with the hepatocarcinogen 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB) causes the appearance in the liver cytosol of a 60 kilodalton oncofetal protein. The appearance of this factor occurs within 40 h of treatment and coincides with the increase in the amount of rapidly labeled RNA released from nuclei in a reconstituted cell-free system. Cross-over experiments show that this increase is due to an enhanced transport capacity of the cytosol. The 60 kilodalton RNA transport factor is also present in the cytosol of tumor cells. Addition of the 60 kilodalton factor to normal liver cytosol causes the transport of repetitive RNA sequences similar to those transported from liver nuclei to tumor cell cytosol and those transported to the tumor cell cytoplasm in vivo. This factor modifies nuclear RNA restriction, at least in part, by eliciting the transport of repetitive RNA normally retained within the nucleus of the normal cell.

The effect of the non-steroidal inhibitor of prostaglandin synthesis, flurbiprofen, has been studied on a transplanted mouse mammary tumour of spontaneous origin. Daily administration of 2.5 mg/kg flurbiprofen signficantly reduced the growth of this NC adenocarcinoma transplanted subcutaneously into the right flank of WHT/Ht mice. Histological examination showed that primary tumours excised at 3 weeks from treated mice tended to have a more prominent lymphocytic infiltration. There was a non-significant tendency for mice treated with flurbiprofen alone to survive longer after tumour excision and to develop less local metastases, although scar recurrences seemed unaffected. The effect of adjuvant chemotherapy and local radiotherapy on local metastases and survival time tended to be greater when flurbiprofen was also given. This work supports and extends the basis for prospective clinical trials to evaluate the role of prostaglandins in human cancer and the value of prostaglandin synthesis inhibitors in cancer therapy.