I. Introduction

The H1, or linker, histones are a well characterized, multivariant family of small basic proteins that play a major role in chromatin organization (Ramakrishnan, 1997 and Widom, 1998). They bind to chromatin with a high affinity (Mamoon et al, 2002) and at a ratio of up to one or more per nucleosome (Widom, 1998; Simpson, 1978). Recently, it was demonstrated that H1 histone to which the large green fluorescent protein (GFP), is fused at its carboxy-terminal domain, can enter the nucleus, where it appears to bind nucleosomes normally (Lever et al, 2000; Misteli et al, 2000). Experiments conducted with the H1-GFP revealed that H1-GFP binding to chromatin is quite dynamic with a residence time on the order of a minute. These observations suggest that the H1 linker histones would be ideal candidates for construction of fusion proteins that will enter cell nuclei. A potentially useful protein to deliver to the nucleus would be a nuclease to act as a cytotoxic agent. Due to the chromatin binding properties of H1 histone, using it to direct a nuclease to the nucleus of a living cell could well prove to have sufficient cytotoxicity as to be of clinical usefulness.

The nuclease domain of the type IIs FokI restriction-modification enzyme has been used as a source of a nuclease for the construction of chimeric restriction enzymes (Kim and Chandrasegaran, 1994; Huang et al, 1996; Kim et al, 1996, 1997, 1998; Kim and Pabo, 1998). The type IIs FokI restriction-modification enzyme was originally characterized by Kita et al, in 1989. It is useful for the construction of chimeric restriction enzymes because its recognition domain is separate from its cleavage domain. Several chimeric restriction enzymes have been engineered in which the DNA recognition domain of one protein has been fused to the endonuclease domain of FokI. These reports include the linking of Drosophila Ultrabithorax (Ubx) homeodomain to the cleavage domain of FokI restriction endonuclease (Kim and Chandrasegaran, 1994). This group also reported the creation of a novel site-specific endonuclease by linking the N-terminal 147 amino acids of yeast Gal4 to the cleavage domain of FokI endonuclease (Kim et al, 1998). The fusion protein was found to be active and under optimal conditions, bound to a 17 bp consensus DNA site and cleaved near this site. Subsequent reports described the engineering of a zinc-finger-FokI restriction endonuclease and characterization of its DNA cleavage specificities (Huang et al, 1996; Kim et al, 1996, 1997 and Kim and Pabo, 1998). These chimeric nucleases have also been used in several application related studies. For example, a zinc-finger-FokI nuclease wasused to cause DNA cleavage and mediate homologous recombination between DNA sequences in Xenopus laevis oocyte nuclei (Bibikova et al, 2001). Similarly, the zinc-finger-FokI chimeric nuclease was also used to stimulate gene targeting in human cells (Porteus and Baltimore, 2003) and in Drosophila (Bibikova and Beumer, 2003). Beyond the use of FokI chimeras to stimulate homologous recombination, the cytotoxicity of these chimeras has yet to be evaluated. The linkage of the FokI nuclease domain to H1 would generate a potent nuclease with superior nuclear localization affinity and avid DNA binding potential. Furthermore, since the desired outcome is to generate lethal levels of DNA cleavage, the low sequence preference of H1 (Wellman et al, 1994; Wellman et al, 1999; Mamoon et al, 2002; Renz, 1975 and Marekov and Beltchev, 1978) with its relatively short residence time (Lever et al, 2000; Misteli et al, 2000) makes it an ideal candidate to reputably deliver a nuclease to the proximity of DNA. In this paper we demonstrate the efficacy of this idea by using a transient transfection system in which an H1 linker histone is used to deliver sufficient nuclease activity to cells, to be lethal.

II. Materials and methods

A. Cloning of the vector DNA constructs

The 588 bp FokI endonuclease domain was amplified by Polymerase Chain Reaction (PCR) of the plasmid, pUC 19/FokI (ATCC) using two primers, UNcoIFokI (5'-CCATGGGTGTGACTAAGCAAC-3') and DBamHIFokI (5'-GGATCCATTAAAGTTTATCTCGCC-3') [all primers were from Integrated DNA Technologies, Coralville, Iowa, U.S.A.] carrying Nco I and BamH I sites respectively. The PCR cycle was [(95∞C, 5 minutes; 95∞C, 1 minute; 46∞C, 1 minute; 72∞C, 1 minute)x5 cycles and (95∞C, 1 minute; 54∞C, 1minute; 72∞C, 1minute)x15cycles]. The Nco I and BamH I (all cloning enzymes were from New England Biolabs) digested PCR product and 4904 bp pBSIIKS(-)/H1∞ (kindly given by Brown, D.T., University of Mississippi Medical Center, Jackson, Mississippi 39216, U.S.A.) vector DNA were ligated to form the 4144 bp pBSIIKS(-)/H1∞-FokI. Next, the cohesive and blunt ends generated by the sequential Stu I and BamH I digestion of the 3578 bp PBSIIKS (-)/H1∞and the 4144 bp pBSIIKS(-)/H1∞-FokI INT vectors were ligated in a two-step reaction. Using this scheme, the sequence of H1∞ was fused in frame (with the insertion of a GCC triplet coding for alanine between the junctions of the two sequences) to the cleavage domain of FokI, to form the 4184 bp pBSIIKS(-)/H1∞-FokI. For the subcloning of FokI endonuclease, the 4184 bp pBSIIKS(-)/H1∞-FokI was digested with Nco I and BamH I enzymes. This yielded the 2988 bp pBSIIKS(-) vector, 588 bp H1∞ and 602 bp FokI cleavage domain. The 2988 bp pBSIIKS(-) vector and 588 bp FokI insert DNA were ligated to form the 3596 bp pBSIIKS(-)/FokI. The construction of vector DNA sequences used for transfection, immunofluorescence, western blot and protein purification experiments was done by similar strategies (Dissertation, Alam Naved, University of Mississippi Medical Center, Jackson, Mississippi 39216, U.S.A). The primary constructs pertinent to this work are shown in Figure 1.

B. Transient transfection of the mouse cells with DNA sequences

Balb/c 3T3 mouse fibroblasts (clone A31) from American Type Cell Culture (Manassas, Virginia, U.S.A.) were grown and maintained as previously described (Brown et al, 1996). Approximately 5.5x10⁵cells plated in a 60 mm culture dish (Corning Inc., Corning, New York, U.S.A.) were grown to 70% confluence. 2 μg of plasmid DNA was suspended in Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, California, U.S.A) [without serum and antibiotics] to a total volume of 150 μl. 15 μl of the Polyfect transfection reagent (Qiagen Inc., Valencia, California, U.S.A.) was added to the DNA solution and the DNA-reagent solution was mixed by pipeting up and down 5 times. The mixture was incubated at room temperature for 10 minutes. During the incubation period, cells were washed twice with fresh media to remove dead cells. Three ml of the media was added to cells. After the incubation period, 1ml of media was added to the DNA-reagent solution and it was mixed by pipeting twice. The DNA solution was then added to the cells with gentle shaking of the culture dish. The cells were incubated for expression of the recombinant gene and the transfectants were analyzed between 24-72 hours post-transfection (PT).

C. Staining of the mouse fibroblasts with calcein-AM and ethidium-homodimer cell viability indicator dyes

The cells transfected in single-well Lab-Tek II Chamber Side System (Nalgene Nunc Int., Rochester, New York, U.S.A.) were washed twice with PBS, 48 hours PT and the chamber well was removed to prepare the cells for dye staining. A combination of 2 μM calcein-AM (CN-AM) and 4 μM ethidium homodimer (ET-HD) viability-indicator dyes (Live/Dead Cell Viability Assay Kit from Promega, Madison, Wisconsin, U.S.A.) was used to stain the cells and they were covered with a cover-slip for observation. A fluorescence microscope equipped with a low magnification Nikon Zeiss (Melville, New York, U.S.A.) lens was used to visualize the cells within 5 minutes of staining them with the dyes. The CN-AM and ET-HD fluorescence was observed using a Fluorescein isothiocyanate (FITC) and a Texas Red filter respectively.

D. Immunofluorescence staining of the mammalian transfectants

The cells transfected in single-well Lab-Tek II Chamber Side System (Nalgene Nunc Int., Rochester, New York, U.S.A.) were washed twice with phosphate buffer saline (PBS), 48 hours PT and the chamber wells were removed for further treatment. Following cell fixation in 4% formaldehyde/PBS for 10 minutes, the cells were treated with 0.5% Triton-PBS for 5 minutes to permeabilize the cell membranes. The cells were washed thrice with PBS and non-specific binding sites for the antibody were reduced by incubating the cells in 10% goat sera/PBS for 30 minutes at 37∞C. The cells were then incubated with a 1:100 dilution of anti-myc antibody (c-Myc 9E10 from Santa Cruz Inc., Santa Cruz, California, U.S.A.) for 1hour at 37∞C. Thereafter, the cells were washed twice with PBS and they were incubated with a 1:400 dilution of anti- mouse IgG₁-FITC antibody (Santa Cruz Inc., Santa Cruz, California, U.S.A.) from goat at 37∞C. After 1 hour, the cells were washed thrice with PBS and treated with 300 nM solution of 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) dye (Molecular Probes, Carlsbad, California, U.S.A.) for 5 minutes to stain the DNA of nuclei. A drop of anti-fade reagent (Molecular Probes, Carlsbad, California, U.S.A.) was added to the slide to preserve the fluorescence intensity and the cells were covered with a cover-slip. DAPI staining of the cells was observed with a DAPI filter using a fluorescence microscope while the fluorescence from the myc-tagged protein was observed with a FITC filter.

E. Western blot analysis of whole cell lysates from the mammalian transfectants

Cells were grown and transfected in 75 cm² culture flasks and were washed thrice with PBS, 72 hours PT and harvested using a cell scraper. They were suspended in 1ml lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 2 mM EDTA, complete EDTA-free protease inhibitor cocktail tablet] for lysate preparation. The cell lysate was sheared by passing through a 23G syringe, centrifuged at 10,000 g for 10 minutes and the protein-rich supernatant was collected. An aliquot of the lysate was resolved by electrophoresis on a 10% SDS-PAGE gel. The proteins were transferred to a nylon membrane (Biorad, Hercules, California, U.S.A.) and non-specific binding sites for the antibody were reduced by incubating the membrane in 10% goat sera/PBS for 1 hour at room temperature. The membrane was then incubated with a 1:100 dilution of anti-myc antibody (c-Myc 9E10 from Santa Cruz. Inc., Santa Cruz, California, U.S.A.) for 1 hour at room temperature. After three washes with PBS, the membrane was further incubated with a 1:5000 dilution of anti-mouse IgG₁-HRP antibody (Pierce Inc., Rockford, Illinois, U.S.A.) from goat at room temperature for 1 hour. Detection of the myc-tagged protein was performed using the recommended protocol of Pierce Inc. and the signal was recorded on Eastman Kodak (Rochester, New York, U.S.A.) Biomax ML X-ray film.

F. Quantitative determination of the cell viability of transfectants by propidium iodide staining

The cells transfected in 60 mm culture dishes were harvested 72 hours post transfection, washed twice with PBS and suspended in 1 ml of fresh PBS. The cells were stained with (100 μg/ml) propidium iodide (PI) dye [Sigma Aldrich Corp., St. Louis, Missouri, U.S.A] and analyzed by flow cytometry within 5-10 minutes of dye staining. 10,000 events were captured by a Coulter SC500 (Beckman Coulter, Fullerton, California, U.S.A.) flow cytometer. Staining was quantitated on a log scale with respect to forward light scatter of the cells. The background intensity of PI staining was substracted from the final calculation for the determination of cell viability for each transfectant. The necrotic control cells were prepared by treatment with methanol for 10 minutes at -20∞C.

G. Annexin V and propidium iodide staining of the mammalian transfectants

The cells transfected in 60 mm culture dishes were washed thrice with PBS, 72 hours post-transfection and harvested in the same solution with the aid of a cell scraper. They were collected by centrifugation at 10,000 g for 30 seconds at 4∞C. The cell pellet was re-suspended at 1x10⁶ cells/ml in 1X annexin-binding buffer (all assay reagents were from Molecular Probes, Carlsbad, California, U.S.A). Next, 5 μl of annexinV alexa fluor 488 dye and 1μl (final concentration of 100 μg/ml) of PI dye were added to a 100 μl aliquot of the cells. The cells were then incubated in dark for 15 minutes. 400 μl of 1X annexin binding buffer was added to the cells and they were mixed briefly and stored at 4∞C. The cells were analyzed by flow cytometry within 10 minutes of the protocol's final step. A population of cells was induced to undergo apoptotic death by a combination of serum-starvation for 72 hours and treatment with the apoptosis-inducing drug, camptothecin (10 μM in dimethyl sulfoxide) [both reagents were from Invitrogen Inc., Carlsbad, California, U.S.A.] for 12 hours (inclusive of the 72 hours serum-starvation period) before the harvesting of cells for analysis.

H. In vitro assay for the cleavage of DNA by purified proteins

Rosetta DE3 plys S cells (Novagen, San Diego, California, U.S.A.) were co-transformed with various pET16b vector constructs along with pACYC/lig (kindly provided by Dr. S. Chandrasegaran, Department of Environmental Health Sciences, The John Hopkins University, Baltimore, Madison, 21205, U.S.A.). A single colony of transformed cells was used for the preparation of protein for a 1L culture. Production of the protein from bacteria and its purification using metal-chelate chromatography was performed according to instructions from the fifth edition of the Qiagen (Valencia, California, U.S.A.) manual. Plasmid pUC 18 DNA (New England Biolabs, Ipswich, Massachusetts, U.S.A.) was incubated with equal concentrations of E.coli produced proteins in DNA cleavage buffer [75 mM KCl, 10 mM MgCl₂, 10 mM Tris-Cl; pH 8.0, 3 mM DTT, 5% glycerol, 100 mg/ml of E. coli tRNA and 50 μg/ml bovine serum albumin]. The digestion reaction was for 4 hours at 37∞C followed by analysis of the products by electrophoresis on ethidium bromide (0.5 mg/ml) stained 0.7% agarose gel.

III. Results

A. The introduction of H1⁰-FokI chimeric DNA in mouse fibroblasts is cytotoxic

We transiently transfected mouse 3T3 fibroblasts with the sequences coding for the H1⁰-FokI hybrid (Figure 1, materials and methods) in order to drive abundant expression of the H1⁰-FokI hybrid off of the strong CMV promoter. Viability was assessed by staining of the cells with a combination of calcein-AM and ethidium homo-dimer dyes. This dual color assay, which discriminates between live and dead cells, was used to observe differences in staining pattern of H1∞-FokI transfected and control cells.

As shown in Figure 2, it is evident that by 72 hours, cells that are transiently transfected with the H1⁰-FokI hybrid construct show significantly more cell death than cells mock-transfected or transfected with control vectors carrying only the H1⁰ or the FokI cleavage domain. The only cells showing significant cell death other than those transfected with the H1⁰-FokI hybrid construct were the control cells killed with methanol.

B. The H1∞-FokI protein re-localizes from the cytoplasm to the nucleus in the transfected cells

To verify that the H1⁰-FokI hybrid localized to the nucleus as predicted and as indicated by the cytotoxic effect of transfection with an H1⁰-FokI hybrid expressing vector, we subcloned the H1⁰-FokI hybrid, the H1⁰ and the FokI nuclease coding regions into a pcDNA3.1(-)myc-HisA (Invitrogen Inc. Carlsbad, California, U.S.A) vector such that they would carry a myc tag on their carboxy termini upon expression (materials and methods). Mouse 3T3 fibroblasts were transiently transfected and the location of the myc-tagged proteins were visualized by immunofluorescent-staining to the myc antigen as described in materials and methods. These results are shown in Figure 3. As expected, H1⁰ localizes strictly to the nucleus. The FokI nuclease domain by itself contains no DNA binding, recognition or nuclear localization capacity and accordingly remains in the cytoplasm. The H1⁰-FokI hybrid is as predicted also found in the nucleus although some is also seen in the cytoplasm in some of the cells.

C. Western blot analysis of cell lysate from transfected mouse cells

A western blot of extracts of these transfectants, using antibodies to the myc tag, demonstrates that the transiently expressed proteins are the expected full length (Figure 4) and the cell viability results are not due to the breakdown of any of the transfectant products. The myc-tagged H1∞ protein migrates with an apparent mobility of 37 kD although the protein has a calculated molecular weight of approximately 22 kD. This altered mobility on SDS-PAGE gels is well described (Welch and O'Rand, 1990; Kasinsky et al, 2001; Nicholson et al, 2004) and is due to the presence of highly positively charged lysine and arginine residues in the carboxy terminal tail. Likewise, the myc-H1∞-FokI protein exhibits a mobility deviation from the predicted molecular weight of roughly 44 kD. The myc-FokI protein, lacking the H1∞ component, migrates as expected on the gel.

can carry the relatively large green fluorescent protein linked to their carboxyl terminal tail into the nucleus and bind to chromatin appropriately (Lever et al, 2000 and Misteli et al, 2000). We thought that it was also likely that the carboxyl tails, which lie along the linker DNA (Zhou et al, 1998; Pruss and Wolffe, 1993), would position the nuclease such that it could readily cut the linker DNA. The in vivo turnover of H1 binding (Lever et al, 2000; Misteli et al, 2000) is also such that it would continue to come off and rebind to chromatin to further enhance the cleavage of the nuclear DNA. It seemed likely that such a chimera could cleave a sufficient amount of DNA such that the cellular repair systems are overwhelmed and the cells would die even if they had become resistant to apoptotic triggers (Sellers and Fischer, 1999; Gatti and Zunino, 2005). Although we demonstrate that H1⁰-FokI probably induces apoptosis, we have no reason to believe that the chimeric protein cannot still kill cells that have become resistant to apoptotic triggers; presumably it would eventually kill all cells through persistent cleavage of the nuclear DNA.

Because the experiments reported here were intended to be strictly a proof of principle, to see if H1 could be used to carry a nuclease into the cell and sufficiently fragment the nuclear DNA such that cell death would ensue, we chose transient transfection as the way to deliver the gene expressing the H1-nuclease chimera. Transient transfection is typically inefficient and its efficiency varies greatly, depending on cell type, vector and transfection protocol (Sambrook et al, 1989). We observed approximately 70% transfection efficiency and therefore did not expect to get 100% killing of the cells upon transient transfection with the H1⁰-FokI expression construct. Also, not all of the cells that become transfected will necessarily express sufficient amounts of H1⁰-FokI to result in cell death. We nevertheless, saw a killing efficiency of 63% (Figure 5) for the cells transfected with the H1⁰-FokI expressing construct. With methods of gene delivery that result in higher copy numbers of exogenous genes, H1⁰-FokI will likely yield a 100% kill rate.

Although our data indicate that H1⁰-FokI transfected cells kill via an apoptotic mechanism, this does not mean that it would not kill cells that are resistant to apoptotic triggers. Except for the necrotic control cells generated by treatment with methanol, all of the cells that died probably did so by an apoptotic mechanism. It is known that transient transfection with plasmids in itself can kill cells and that this death in many cell lines is apoptotic (Rodriguez and Flemington, 1999).

H1 histones themselves have recently been shown to be potentially lethal to cells, (Tsoneva et al, 2005). In this study, the H1 was abundantly delivered by electroporation and the mechanism of killing was believed due to effects on the mitochondria. When delivered via a gene expression system, we observe no lethality due to H1 alone. This is in agreement with previous studies that relied on overexpression of H1 histones (Brown et al, 1996, 1997; Gunjan et al, 1990; Gunjan and Brown, 1999). We cannot exclude the possibility that the effect of H1 alone on viability is cell-type specific; electroloaded H1 did not have the same killing effect on non-transformed cells as it did on transformed cells (Tsoneva et al, 2005). However, we have not seen a lethality that can be attributed to H1 alone (Brown et al, 1996, 1997; Gunjan et al, 1990; Gunjan and Brown, 1999). However, we typically select permanent transfectants and the selection process may eliminate or select for resistance to H1 toxicity. We also cannot eliminate the possibility that some H1 variants are lethal. We have been unsuccessful in the selection of some permanent transformants of H1 variants or mutants that can be induced to overproduce significant amounts of the particular H1 type (unpublished data).

It has been recently demonstrated that upon induction of apoptosis with agents that generate double-stranded breaks in DNA, such as with X-rays or with etoposide, the histone variant H1.2 (H1c) may be the apoptotic inducer (Konishi et al, 2003). As with the electroloaded H1s (Tsoneva et al, 2005), it appears to elicit a response through an interaction with mitochondria causing the release of cytochrome C. Overall, it appears that transfection with an H1 chimeric nuclease is a much more controllable method of killing cells; in situ cell specificity can be obtained by incorporating the H1-FokI gene into a viral vector that has been engineered to be cell type selective.

Acknowledgments

We thank Dr Susan Wellman for critical reading of the manuscript. We also thank Dr. Robert Lewis, Kevin Beason and Susan Touchstone for Fluorescent Activated Cell Sorter analysis.

References

Arends MJ, Morris RG and Wylie AH (1990) Apoptosis: The role of endonuclease. Am J Pathol 136, 593-608.

Bibikova M, Beumer K and Trautman JK (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764.

Bibikova M, Carroll D and Segal DJ (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21, 289-297.

Brown DT, Alexander BT and Sittman DB (1996) Differential effect of H1 variant overexpression on cell cycle progression and gene expression. Nucl Acids Res 24, 486-493.

Brown DT, Gunjan A, Alexander BT and Sittman DB (1997) Differential effect of H1 variant overproduction on gene expression is due to differences in the central globular domain. Nucl Acids Res 25, 5003-5009.

Gatti L and Zunino F (2005) Overview of tumor cell chemoresistance mechanisms. Methods Mol Med 111, 127-148.

Gunjan A, Alexander BT, Sittman DB and Brown DT (1990) Effects of H1 histone overexpression on chromatin structure. J Biol Chem 274, 37950-37956.

Gunjan A and Brown DT (1999) Overproduction of histone H1 varaints in vivo increases basal and induced activity of the mouse mammary tumor virus promoter. Nucl Acids Res 27, 3355-3363.

Huang B, Li CJQ and Tsai MW (1996) Sp1ase: A new class IIs zinc finger restriction endonuclease with specificity for Sp1 binding sites. J Prot Chem 15, 481-489.

Kasinsky HE, Lewis JD, Dacks JB and Ausio J (2001) Origin of H1 linker histones. Faseb J 15, 34-42.

Kim YG, Cha J and Chandrasegaran S (1996) Hybrid restriction enzymes: Zinc finger fusions to FokI cleavage domain. Proc Natl Acad Sci 93, 1156-1160.

Kim YG and Chandrasegaran S (1994) Chimeric restriction endonuclease. Proc Natl Acad Sci 91, 883-887.

Kim YG, Kim PS, Hebert A and Rich A (1997) Construction of a Z-DNA-specific restriction endonuclease. Proc Natl Acad Sci 94, 12875-12879.

Kim JS and Pabo CS (1998) Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc Natl Acad Sci 95, 2812-2817.

Kim YG, Smith SJ, Durgesha M and Chandrasegaran S (1998) Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain. Biol Chem 379, 489-495.

Kita K, Kotani H, Sugisaki H and Takanami M (1989) The FokI restriction-modification system. J Biol Chem 264, 5751-5756.

Konishi AM, Shimizun S, Hirota J, Takao T, Fan Y, Matsuoka Y, Zhang L, Yoneda Y, Fujii Y, Skoultchi AI and Tsujimoto Y (2003) Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks. Cell 114, 655-656.

Lever MA, Th'ng JPH, Sun X and Hendzel M (2000) Rapid exchange of histone H1.1 on chromatin in living cells. Nature 408, 873-876.

Mamoon N, Song Y and Wellman SE (2002) Histone H1(0) and its carboxyl-terminal domain bind in the major groove of DNA. Biochem 41, 9222-9228.

Marekov LN and Beltchev B (1978) Preferential binding of phosphorylated histone H1 to AT rich DNA. Int J Biol Macromol 3, 145-147.

Misteli T, Gunjan A, Hock R, Bustin M and Brown DT (2000) Dynamic binding of histone H1 to chromatin in living cells. Nature 408, 877-881.

Moffatt J, Hashimoto M, Kojima A, Kennedy DO, Murakami A, Koshimizu K and Yuasa IM (2000) Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation. Carcinogenesis 21, 2151-2157.

Nicholson JM, Wood CM, Reynolds CD, Brown A, Lambert SJ, Chantalat L and Baldwin JP (2004) Histone Structures: Targets for modifications by molecular assemblies. Ann N.Y. Acad Sci 1030, 644-655.

Porteus H and Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763.

Pruss D and Wolffe AP (1993) Histone-DNA contacts in a nucleosome core containing a Xenopus 5S rRNA gene. Biochem 32, 6810-6814.

Ramakrishnan V (1997) Histone structure and the organization of the nucleosome. Annu Rev Biophys Biomol Struct 26, 83-112.

Renz M (1975) Preferential and cooperative binding of histone H1 to mammalian chromosomal DNA. Proc Natl Acad Sci 72, 733-736.

Rodriguez A and Flemington EK (1999) Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies. Anal Biochem 272, 171-181.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, New York.

Sellers WR and Fischer DE (1999) Apoptosis and cancer drug targeting. J Clin Inves 104, 1655-1661.

Simpson R (1999) Structure of the chromatosome, a chromatin particle containing 160 bp of DNA and all histones. Biochemistry 17, 5524-5531.

Tsoneva I, Nikolova B, Georgieva M, Guenova M, Tomov T, Rols MP and Berger MR (2005) Induction of apoptosis by electrotransfer of positively charged proteins as cytochrome C and histone H1 into cells. Biochim Biophys Acta 1721, 55-64.

Welch JE and O'Rand MG (1990) Characterization of a sperm-specific nuclear autoantigenic protein. II. Expression and localization in the testis. Biol Reprod 43, 569-578.

Wellman SE, Sittman DB and Chaires JB (1994) Preferential binding of H1e histone to GC-rich DNA. Biochem 33, 384-388.

Wellman SE, Song Y and Mammon NM (1999) Sequence preference of mouse H1∞ and H1t. Biochem 38, 13112-13118.

Widom J (1998) Structure, dynamics and function of chromatin in vitro. Annu Rev Biophys Biomol Struct 27, 285-327.

Wylie AH, Arends MJ, Morris RG, Walker SW and Evan G (1992) The apoptosis endonuclease and its regulation. Sem Immunol 4, 389-398.

Yakovlev A.G and Faden AI (2001) Capsase-dependent apoptotic pathways in CNS injury. Mol Neurobiol 24, 131-144.

Yeh CT and Yen GC (2005) Effect of sulforaphane on metallothionein expression and induction of apoptosis in human hepatoma HepG2 cells. Carcinogenesis 26, 2138-2148.

Zhou YB, Gerchman SE, Ramakrishnan V, Travers AA and Muyldermans SV (1998) Position and orientation of the globular domain of linker histone H5 on the nucleosome. Nature 395, 402-405.

Research Article

Received: 13 April 2006; Accepted: 19 April 2006; electronically published: May 2006

I. Introduction

References