In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.

The binding of all known linker histones, named H1a through H1e, including H1 0 and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8 -16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1 0 , completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270 -360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.H1 histones are a heterogeneous group of at least five subtypes with closely related but nonetheless different primary structures (1, 2). Two further H1 subtypes are known: the histone H1 0 , which is found in nonreplicative tissues (3, 4) and in rapidly proliferating cells (5), and the testis-specific histone variant H1t (6). The various linker histones containing a globular central region flanked by highly basic and hydrophilic C-and N-terminal tails (7,8) bind to the nucleosome and promote the organization of nucleosomes to a higher order structure (9, 10).There is evidence that histone H1 may interact differently with transcriptionally active and inactive regions of chromatin (11). Linker histones are also thought to modulate nucleosome position (12, 13) and to influence replication efficiency in vitro (14).The presence of this large number of various H1 histone subtypes and their possible posttranslational modifications, such as phosphorylation (15), make it very probable that H1 histones play numerous structural and functional roles in chromatin. Until now, no specific role for the various variants has been established although Kaludov et al. (16) showed that the mouse histone H1b binds preferentially to a regulatory sequence within a mouse H3.2 replication-dependent histone gene. Previous analysis of the structural role of H1 histones demonstrated that three subfractions of H1 histones differ in their effectiveness in condensing DNA fibers into ordered aggregates (17) and that histone subtype H1t, compared with other subtypes, dif...

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogenactivated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFa-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmkor zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

This study presents first data with respect to early and late in vivo response on a strontium functionalized titanium surface comprising a nanotopography manufactured by a magnetron sputtering process. We investigated different osseointegration stages of screw-shaped implants with dental implant geometries in a rabbit femur model observing beneficial effects of the functionalized surface on bone-to-implant contact and bone formation caused by tailored release of the bone anabolic strontium. Histomorphometrical data revealed that a functionalized titanium surface with controlled liberation of strontium accelerates osseointegration while spectrometry measurements did not indicate a potential systemic effect of this osteoinductive agent and could thus have impact on modifications of medical implant devices.

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.

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Gemcitabine (2≺,2≺difluoro‐2≺deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen‐sensitive tumor cell line LNCaP and the androgen‐insensitive cell lines PC‐3 and DU‐145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co‐administration of 10–100 μM of its natural analogue deoxycytidine. In view of a future clinical application of this anti‐tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic‐macrophagic progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic‐macrophagic progenitor cells required a concentration of 9 nM. Co‐administration of deoxycytidine to gemcitabine‐treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic‐macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma. © 1996 Wiley‐Liss, Inc.