Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine-pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.

Loss of amplified c-myc genes in the spontaneously differentiated HL-60 cells.

Amplification of the c-myc gene in the human promyelocytic leukemia cell line HL-60 is considered to be one of the major causes of its malignant phenotype. It is also well known since the establishment of the cell line that a culture of HL-60 cells contains a small but fixed percentage of spontaneously differentiated cells. We show that the spontaneous differentiation could be a result of extensive losses of amplified c-myc genes by the findings: (a) the spontaneously differentiated HL-60 cells express Mac-1 (CR3, CD11b/CD18) antigen, irreversibly stop the uptake of [3H]thymidine, and die by apoptosis; (b) these cells, when isolated, and when the copy number of c-myc genes is precisely quantitated, show extensive losses of c-myc genes; and (c) low concentrations of hydroxyurea increase the percentage of spontaneously differentiated cells in which the number of c-myc genes is further decreased. A simple theoretical consideration suggests that an active elimination process(es) must be operating besides the stochastic losses of the extrachromosomally amplified c-myc genes by unequal partition at mitosis.

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions.

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.

A gene coding for a zinc finger protein is induced during 12-O-tetradecanoylphorbol-13-acetate-stimulated HL-60 cell differentiation.

ETR103 cDNA was cloned as an immediate early gene in the course of macrophagic differentiation of HL-60 cells stimulated by TPA (12-O-tetradecanoylphorbol-13-acetate). The induction by TPA was immediate-early (within 30 min) and transient. This gene was not induced by vitamin D3 or by retinoic acid, which stimulates differentiation of HL-60 cells to the monocytic or granulocytic lineage, respectively. The ETR103 mRNA was induced by TPA in lymphoid or myeloid leukemia cell lines of several maturation stages. The induction by TPA seems to proceed by a protein kinase C-mediated mechanism, on the basis of the results obtained by using protein kinase C inhibitor (H-7), protein kinase C activator (diC8), and an activator of protein kinase A (dibutyryl cAMP). Okadaic acid, an inhibitor of protein phosphatases, also induced the ETR103 mRNA expression. The nucleotide sequence of the ETR103 cDNA reveals that ETR103 encodes a human zinc finger-containing transcription factor identical to Egr-1 and 225, which is homologous to mouse Egr-1, Zif/268, Krox-24, and TIS8, or to rat NGFI-A.

Determination of transcriptional activities of typical gene promoters in HL-60 cells.

We developed a highly sensitive procedure for assaying chloramphenicol acetyltransferase (CAT) enzyme activity in extracts of eukaryotic cells transfected with the CAT gene expression vector, by modification of the partition extraction procedure described by Sleigh [Anal. Biochem. 156, 251-256 (1986)]. The sensitivity of the new method was improved 100-fold on commercial purified enzyme. In routine measurements with cell extracts a CAT activity as low as 1.3 x 10(-4) unit could be measured within an error of less than 30%. The CAT enzyme expressions in undifferentiated human promyelocytic leukemic cell line HL-60 from typical gene promoters could be measured by the new method and compared to select a stronger promoter. Similar measurements were made with more mature monocytic THP-1 cells to evaluate the change in the promoter activity with cell maturation. Differentiation induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) activated transcription from the human immunodeficiency virus (HIV) promoter about 10-fold in HL-60 cells, as expected, but the level was less than that in untreated THP-1 cells. In addition, a similar activation was observed in THP-1 cells as well.

Interplay between differentiation and cell cycle arrest at G1 in phorbol diester-treated human myelocytic leukemia cells.

The cell cycle arrest at G1 of myelocytic leukemia cells by incubation with phorbol diesters was examined in HL-60 cells and more mature THP-1 cells. We found: (1) induction of phenotypic parameters of differentiation prior to G1 arrest; (2) a long induction period in HL-60 cells compared to zero in THP-1 cells; and (3) strong inhibition of G1 arrest with a protein kinase C inhibitor in THP-1 cells. We conclude, from these lines of evidence and some additional information, that differentiation induces G1 arrest. At present, the most plausible causative molecule is Mac-1.

Co-localization of mitochondrial and double minute DNA in the nuclei of HL-60 cells but not normal cells.

In an attempt to isolate genes located on double minute (Dmin) DNA in HL-60 cells, we prepared DNA probe from purified micronuclei. Micronucleation was induced in HL-60 cells by treatment with hydroxyurea. Screening of a cDNA library unexpectedly produced a number of clones containing mitochondrial DNA (mtDNA) sequences. Here, we show that amplified mtDNA sequences were localized in nuclei and micronuclei of HL-60 and COLO 320DM cells, but not in nuclei of WI-38 normal human fibroblasts or peripheral blood T-cells. To unequivocally demonstrate the presence of mtDNA inside of nuclei and micronuclei, we obtained tomographic fluorescence in situ hybridization (FISH) images of mtDNA by confocal microscopy of consecutive sections of paraformaldehyde (PFA)-fixed material. We also located mtDNA in nuclear buds and purified micronuclei. Dmin DNA and mtDNA were always located at similar sites. The mechanisms of nuclear retention of mtDNA and Dmin DNA and the resulting influence on tumorigenesis are discussed.

Mechanism and role of cooperative binding of bacteriophage fd gene 5 protein to single-stranded deoxyribonucleic acid.

The highly cooperative binding of fd gene 5 to single-stranded DNA was studied kinetically by rapid photo-cross-linking and stopped-flow UV absorption measurements. The observed change in absorbance was shown to be due to the binding by direct evidence of rapid photo-cross-linking of the bound proteins to fd DNA. The bimolecular rate constant obtained for the association was 1.6 X 10(10) M-1 s-1 (in terms of the molecular concentration of DNA), which was concluded to be diffusion controlled. The breakdown of cluster complexes on fd DNA was induced by the addition of large excess amounts of short single-stranded DNA. The breakdown took place in about 1 s. The kinetic process of redistribution of dissociated proteins was monitored by rapid photo-cross-linking and subsequent electrophoresis of the cross-linked complex. The dissociated proteins first formed isolated complexes, but later they were again converted into the cluster. The kinetic results showed that the cooperativity originated from the stabilization of the protein-DNA complex by the cluster formation, not from the accelerated association in the cluster formation. This kind of cooperative binding was shown to perform negative feedback control in the cluster formation. On the basis of the kinetic results obtained, we proposed a model for the regulatory role of the fd gene 5 protein in the synthesis of single-stranded fd DNA.

Low Angle, Light Scattering Photometer Model LS-2.

A light scattering instrument capable of routine measurements at angles as low as 9 degrees is described. The reduced scattered intensities can be determined, with use of a vertically polarized incident light, correct to 0.5 ~ 1.0%, unless the scattering of the solution over solvent is less than 10(-5) cm(-1). The correction for the back reflection of the incident beam can be ignored because the light is absorbed by a glass neutral filter before it reaches a glass-air interface. A sample liquid can be brought to a temperature equilibrium in less than 30 min, and the temperature can be kept constant within +/-0.02 degrees C, in the temperature range from 5 degrees C to 100 degrees C. No special optical specification is required in making a cell, and the geometrical correction is simply given by sin-10 correct to +/-0.7%.

Release of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA.

To elucidate the mechanism of sigma release in the transcript by Escherichia coli DNA-dependent RNA polymerase, we obtained the time courses of sigma release and elongation of product RNA by a rapid kinetic technique; transcription was synchronously initiated from A1 promoter on T7 DNA by the addition of four substrates to a stoichiometric mixture of holoenzyme and template DNA, and then quenched by the addition of EDTA. The elongation rate was changed by limiting the concentration of one of four substrates, GTP. At reduced GTP concentration, elongation was decelerated, but the time course of sigma release was unchanged. No connection between sigma release and length of RNA product was found. The results lead to the conclusion that sigma is released depending only on time elapsed after initiation, not on the length of RNA product. We propose a two-step model for sigma release with a rapid triggering and a slow dissociation of about 5 s. This dissociation, the rate-determining step of sigma release, is independent of the rate of elongation.

Identification and characterization of the direct folding process of hen egg-white lysozyme.

Refolding kinetics of hen egg-white lysozyme (HEWL) have been studied by means of the stopped-flow method with guanidinium chloride as the denaturant. We show here that the three-species model U1 in equilibrium or formed from U2 in equilibrium or formed from N (U1 and U2 = unfolded; N = native) now established for pancreatic ribonuclease A is also valid for HEWL on the basis of the following lines of evidence: (1) refolding kinetics outside the transition region are biphasic; (2) dependence of the fractional amplitude for the fast phase on the ratio of the time constants of the two phases agrees with theory; (3) unfolding kinetics outside the transition region are of single phase; (4) direct evidence for the U2 leads to U1 transformation is obtained by double-jump experiments; (5) the time constant of the binding reaction of a substrate analogue, 4-methylumbelliferyl N,-N'-diacetyl-beta-chitobioside, to HEWL molecules during refolding reaction agrees with the time constant of the direct refolding phase U2 leads to N. The characteristic properties of the nucleation-controlled reaction of refolding of small globular proteins are discussed in general. The results of the discussion are used to suggest that the direct folding process is nucleation controlled from the experimental results of the temperature dependence of the refolding rate.

Spectral evidence for a rapidly formed structural intermediate in the refolding kinetics of hen egg-white lysozyme.

For investigation of the conformation of the unfolded species and its role in the refolding kinetics, refolding kinetic measurements were made on hen egg-white lysozyme by using the stopped-flow method at 25 degrees C in the four sets of initial and final folding condition: (1) 4 M guanidinium chloride (GdmCl) and 0.5 M GdmCl; (2) 40% acetic acid (HOAc) and 5% HOAc; (3) 4 M GdmCl and 0.5 M GdmCl-5% HOAc; (4) 40% HOAc and 0.5 M GdmCl-5% HOAc. The kinetic results as measured by absorbance at three wavelengths, 301, 292, 250 nm, agreed with each other and indicated strict biphasic behavior without exception. The kinetic parameters were determined only by the final refolding conditions. The spectral properties of the unfolded species at the end of stopped-flow mixing were investigated by comparing the total kinetic amplitude with the difference between the static absorbance of the native molecule in the final refolding conditions and that of the unfolded molecule in the initial unfolding conditions. The solvent effect was considered in the comparison. It was concluded that the unfolded species assumed a new transient conformation in the mixing process and that the transformation was completed within the mixing time.

Granulocytic differentiation of HL-60 cells, both spontaneous and drug-induced, might require loss of extrachromosomal DNA encoding a gene(s) not c-MYC.

Treatment of HL-60 cells with drugs induces granulocytic differentiation and c-MYC down-regulation that is irreversible and associated with loss of DNase I-hypersensitive sites in c-MYC promoter. The expression of these phenotypes requires a slow process that appears to accompany a loss of c-MYC copies in double minutes via micronuclei. However, the drug treatment induced c-MYC down-regulation very early, though only reversibly. Here we show that we can resolve this paradox by assuming a gene(s) in other extrachromosomal, acentromeric DNA. Treatment with drugs might induce no down-regulation of this gene, but its complete elimination via micronuclei might be necessary and sufficient for the expression of the above phenotypes. Loss of c-MYC copies is unavoidable because the exclusion of extrachromosomal DNAs via micronuclei is at random. This conclusion is based on the observation of a substantial number of c-MYC copies in certain differentiated cells, irrespective of whether the differentiation was induced with drugs or without.

Evolution of large extrachromosomal elements in HL-60 cells during culture and the associated phenotype alterations.

In the HL-60 sublines that were isolated after a long-term continuous culture, abnormally stained or abnormally banded regions on chromosomes replaced extrachromosomal double minutes. The c-MYC gene is amplified in these structures. We followed the c-MYC gene loci during a consecutive passage by using FISH, and have found a large extrachromosomal element (LEE) that preexisted at the earliest passage in a very small fraction of cells. No chromosomal integration of c-MYC sequences was observed in up to 225 passages. The LEEs persistently evolved during culture and were not excluded from the nucleus. In the LEE-positive cells, the spontaneous differentiation was blocked and the granulocytic differentiation that was induced by treatment with dimethyl sulfoxide was reversed by withdrawal of the drug. The c-MYC gene integration into LEEs is unlikely to lead to these phenotypes. The reversibility might be related to the reversible c-MYC down-regulation during the early phase of the drug treatment of HL-60 cells at early cell passages.

Expression of a novel immediate early gene during 12-O-tetradecanoylphorbol-13-acetate-induced macrophagic differentiation of HL-60 cells.

Human promyelocytic leukemia cell line, HL-60, undergoes macrophagic differentiation when it is stimulated with TPA (12-O-tetradecanoylphorbol-13-acetate). We have cloned ETR101 cDNA whose mRNA was induced immediate early (30 min) and transiently by TPA. The mRNA is superinduced by addition of the protein synthesis inhibitor cycloheximide. The sequence of ETR101 cDNA (1826 base pairs) reveals that (i) it will encode a protein of 223 amino acids with a formula molecular weight of 24,200, (ii) the amino acid sequence is highly homologous to mouse chx1 protein whose mRNA was found recently to be enhanced in activated T lymphocytes in response to cycloheximide, (iii) the amino acid sequence is also weakly homologous to jun family gene products, and (iv) in the mRNA 3'-flanking region, there is a unique GUUUG sequence which is complementary to a part of B1 repetitive sequence and may be involved in mRNA degradation. ETR101 mRNA is induced by TPA in a wide variety of leukemia cells including myeloid, T-lymphoid, and B-lymphoid lineages. We have found that this mRNA is also induced by okadaic acid, a protein phosphatase inhibitor, and that TPA or cycloheximide act synergistically with okadaic acid. In addition, the induction is inhibited by protein kinase C inhibitors. Therefore, ETR101 mRNA level is controlled, either directly or indirectly, by protein phosphorylation.

Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.

Expression profile of active genes in granulocytes.

A number of genes active in granulocytes have been intensively studied as to the function of their products and their expression controls. However, the intensities and relative order of these gene activities have not been studied. This report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 3'-directed cDNA library that faithfully represents the mRNA population in the source cells. A significant fraction (20.3% of the total) of the expressed genes in granulocytes consisted of nuclear proteins such as DNA binding proteins, of secretory proteins such as cytokines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing this expression profile with 11 profiles similarly obtained with unrelated human cells/tissues, we discovered 10 novel genes that are likely to act specifically in granulocytes. Comparison of this expression profile with that obtained with granulocytoids widely used as a granulocyte model by inducing a cultured promyelocytic leukemia cell line HL60 showed similarities and dissimilarities of gene expressions.

Survival by Mac-1-mediated adherence and anoikis in phorbol ester-treated HL-60 cells.

During the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence.