Joseph G. Valdez

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase mu Bondapak C18 column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [3H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.

Proteins in the fossil bone of the dinosaur, seismosaurus

Proteins have been successfully extracted from the fossil vertebra of a 150-million-year-old sauropod dinosaur (“Seismosaurus”) recently excavated from the Morrison Formation of New Mexico. HCl and guanidine·HCl extracts of the fossil bone and its sandstone matrix were concentrated, demineralized, and resolved into a number of different protein fractions by reversed-phase high-performance liquid chromatography (HPLC). One of these fractions had the same retention time as collagen. Amino acid analysis (Pico-Tag method) of these fractions confirmed they were proteins. Comparison of the correlation coefficients of the amino acid analyses with that of collagen standards indicated that none of the fractions contained significant amounts of collagen. Similar HPLC profiles were obtained for the HCl extracts of fossil bone and its sandstone matrix suggesting they contained the same proteins. However, different HPLC profiles were obtained when these HCl extracts were dried and reextracted with guanidine·HCl. These different fractions represent proteins unique to the fossil and were not found in the sandstone matrix. These differences were confirmed by amino acid analysis. Such information on fossil bone proteins might provide useful knowledge concerning the evolution of skeletal molecules and the fossilization process. Similar information on the proteins from the geological matrix might provide useful fingerprints for reconstructing ancient environments and for assessing sedimentary rocks for fossil fuel exploration.

Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry

Simultaneous measurement of cellular DNA and RNA content provides information for determination of the functional status of cells and, clinically, for the diagnosis and grading assessment of various tumors. Most current flow cytometric methods are based on resolving the fluorescence emission spectra of dyes that bind preferentially to either type of nucleic acid. However, several monochromatic nucleic acid–binding fluorochromes display resolvable differences in fluorescence lifetime when bound to DNA or RNA. The differences in the lifetime of one fluorescent probe provide an alternate means to distinguish the binding of one probe to these cellular macromolecules and to simultaneously measure their cellular contents.

High-performance liquid chromatography of chromatin histones

A method is described for the rapid analysis of histones by high-performance liquid chromatography on reversed-phase muBondapak columns, containing either octadecylsilane (C18) or cyanopropylsilane (CN) bonded to silica particles packed in either steel columns or Radial-Pak cartridges. A linear gradient progressing from water-acetonitrile (80:20) to water-acetonitrile (40:60) and increasing in acetonitrile concentration at the rate of 10%/h was used to elute the histones at a flow-rate of 1 ml/min for steel columns or 2 ml/min for Radial-Pak cartridges. Two conditions were found to be necessary to achieve histone fractionation: (1) silylation of the active silanol groups on the silica matrix, and (2) 0.1-0.3% trifluoroacetic acid in the eluting solvent. More than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted. The histone fractions were identified by their electrophoretic mobilities in both acid-urea and Triton DF-16 polyacrylamide gels. Histones were eluted from the columns in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less-hydrophobic and more-hydrophobic histone variants). Phosphorylated and acetylated histone molecules were not separated from their unmodified parent molecules. The volatile nature of the water-acetonitrile-trifluoroacetic acid eluting solvent facilitated recovery of salt-free histones from the fractions by direct lyophilization of the column effluents. The best resolution of histone fractions was obtained with the Radial-Pak muBondapak C18 cartridge using 0.3% TFA. However, for analytical studies, the best detection was obtained by using the muBondapak CN steel column. Poorer resolution was obtained by using the non-silica based PRP-1 reversed-phase column, containing a polystyrene-divinylbenzene resin under the same conditions.

Association of G1/S-Phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells

We investigated the time-dependent effects of 8 Gy of gamma radiation on the activities of cyclin-dependent kinases (Cdks) and the incorporation of the thymidine analog bromodeoxyuridine (BrdU) throughout the S phase in Chinese hamster ovary (CHO) cells. The in vitro Cdk activities of immunoprecipitated cyclin E, cyclin A and Cdk2 were reduced about 30% per cell within 0.5-1 h after irradiation, but they recovered at different rates. The kinase activity of the cyclin E-Cdk2 complex recovered first and exceeded the control values by 1.5-2 h after irradiation. Cyclin A-Cdk activities began to recover at 3-4 h after irradiation, and cyclin E/A-Cdk2 activities recovered at intermediate rates. The super-recovery of cyclin E-Cdk2 coincided with the appearance of a small synchronous population of cells entering into S phase, consistent with transient G1-phase delay/recovery regulated by cyclin E-Cdk2, whereas the activities of cyclin A-Cdks (75% cyclin A-Cdk2; 25% cyclin A-Cdc2 when inhibition was maximal) were correlated with rates of total DNA synthesis. Multivariate flow cytometry analyses of BrdU incorporation demonstrated that radiation-induced inhibition of DNA synthesis occurred predominantly within the last quarter of S phase and that the majority of the irradiated cells failed to enter G2 phase for 4-5 h. The recovery of cyclin A-Cdk activities coincided with increased levels of total DNA synthesis and BrdU incorporation into cells within the last quarter of S phase. Western blot analysis demonstrated that levels of Waf1/p21 did not increase during inhibition of cyclin A-Cdks and DNA synthesis in the irradiated p53-mutated CHO cells; however, Cdc2 and Cdk2 exhibited increased levels of phosphotyrosine. The results (1) indicate that the transient G1-phase delay or G1/S-phase checkpoint (Lee et al., Proc. Natl. Acad. Sci. USA 94, 526-531, 1997) is mediated by inhibition of cyclin E-Cdk2 and (2) point to the existence of a radiation-induced S-phase checkpoint located about 75% into S phase involving the inhibition of cyclin A-Cdks by a p53/Waf1-independent pathway in CHO cells.

Preparation of histone variants and high-mobility group proteins by reversed-phase high-performance liquid chromatography

Methods have been developed for the preparation of histone variants and high-mobility group (HMG) proteins by high-performance liquid chromatography (HPLC). The individual HPLC fractions were recovered as a dry powder in 95% yield by direct lyophilization from the column effluent. Perchloric acid-soluble H1 variants and HMG proteins from Chinese hamster cells (line CHO) were separated on a mu Bondapak CN column using a 0-50% linear acetonitrile gradient in water containing 0.2% trifluoroacetic acid (TFA). The proteins were eluted in the following order: HMG-E/G (an HMG-14/17 class proteins from CHO cells), HlO, Hl, HMG-2, and HMG-l. HMG-E/G, Hl, and an unidentified protein were recovered electrophoretically pure. HlO contained contaminants which could be removed by subsequent chromatography on a mu Bondapak C18 Radial-Pak column, but HMG-l and HMG-2 could not be completely resolved. Nucleosomal core histones were fractionated on a mu Bondapak C18 Radial-Pak column using a 30-55% linear acetonitrile gradient containing 0.2-0.3% TFA. They were eluted in the following order: H2B, (LHP)H2A, (MHP)H2A, H4, LHP(H3), and (MHP)H3, (where LHP and MHP refer to less-hydrophobic and more-hydrophobic variants). If the gradient containing 0.3% TFA was interrupted with an isocratic elution at 43% acetonitrile, the H2B, (LHP)H2A, (MHP)H2A, and H4 proteins were completely resolved, thus providing a good preparative method for these proteins. The H2A class of Drosophila histones was also fractionated on a mu Bondapak C18 Radial-Pak column using a 30-35% linear acetonitrile gradient containing 0.2% TFA. Drosophila melanogaster H2A, obtained as a single fraction by chromatography on Biol-Gel P-100, was eluted from the C18 column as three proteins. The order of elution was identified by electrophoresis to be: H2Aox (an oxidized form of H2A), D2 (a Drosophila-specific subtype), and H2A.

Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns

Previous work in our laboratory [Gurley et al. Anal. Biochem. 129, 132-144 (1983)] described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase muBondapak C18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a muBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species.

Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide

Deuterium oxide (D2O) has been shown in previous studies to increase both the fluorescence lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis and conventional and phase-sensitive flow cytometry to compare changes in PI and EB fluorescence intensity and lifetime bound to DNA and fixed Chinese hamster ovary (CHO) cells in the presence of D2O vs. phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a twofold enhancement of fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fluorescence lifetime of HL-60 cells stained with PI or EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifetime of these fluorochromes is sensitive to chromatin configuration in different cells types. Apoptotic subpopulations of HL-60 cells had a significantly reduced fluorescence lifetime compared to nonapoptotic subpopulations. Results indicate that different chromatin states, or differences in the structures of PI and EB, lead to alterations in the fluorescence intensity and fluorescence lifetime of these intercalating probes.

High-performance capillary electrophoresis of histones

A high-performance capillary electrophoresis (HPCE) system was developed for the fractionation of histones. This system involves electroinjection of the sample and electrophoresis in 0.1 M phosphate buffer (pH 2.5) in a 35 cm x 50 micron I.D. coated capillary. Electrophoresis was accomplished in 9 min, separating a whole histone preparation into its components in the following order of decreasing mobility: (MHP) H3, H1 (major variant), H1 (minor variant), (LHP) H3, (MHP) H2A (major variant), (LHP) H2A, H4, H2B and (MHP) H2A (minor variant), where MHP is the more hydrophobic component and LHP is the less hydrophobic component. This order of separation is very different from that found in acid-urea polyacrylamide gel electrophoresis and in reversed-phase high-performance liquid chromatography and, thus, brings the histone biochemist a new dimension for the qualitative analysis of histone samples.