Here we produced a monoclonal antibody (termed as mH2) which specifically recognized an 86-kD subunit of Human DNA helicase II (p86) in HeLa cells. Immunohistochemical analysis of p86 throughout the cell cycle showed that its localization was changed from the nuclei to the cytoplasm, when the cells entered into prophase, and p86 localized diffusely in the cytoplasm during mitosis. Double staining of interphase HeLa cells with mH2 and an antibody, which recognized a component of nuclear pore complex, showed that p86 did not localize at the nuclear envelop in interphase cells.
INTRODUCTION
We generated several strains of monoclonal antibodies against mitotic HeLa cells for studying the molecular identity of substances involved in mitosis (Okubo et al., 1999). Among these, one monoclonal antibody (designated as mH2) revealed the dynamic change of subcellular localization of its antigen between interphase and mitotic HeLa cells. The mH2 specifically recognized a single 86-kD component in HeLa cell extract and its antigen was identified as an 86-kD subunit of human DNA helicase II (p86).
Human DNA helicase II (HDH2) is ATP-dependent DNA unwinding enzyme purified from HeLa cells (Tuteja et al., 1990). HDH2 was first described as Ku autoantigen, to which antibodies are produced in patients with scleroderma polymyositis overlap syndrome (Mimori et al., 1981), and later found in the sera of patients with other rheumatic diseases (Mimori et al., 1986). HDH2 is composed of two subunits, which were called p70 and p86, respectively (Yaneva et al., 1985; Reeves, 1985; Mimori et al., 1986). Both subunits have a leucine zipper in their primary sequences, which explains their ability to associate with each other and to bind DNA (Reeves and Sthoeger, 1989). In the presence of double strand DNA, HDH2 associates with a 350-kD catalytic sub-unit to form the DNA-dependent serine/threonine protein kinase, which phosphorylates a number of transcription factors (Lees-Miller and Anderson, 1991; Dvir et al., 1993; Gottlieb and Jackson, 1993; Suwa et al., 1994).
Using this mH2, we present here the dynamic change of p86 localization throughout the cell cycle in HeLa cells.
MATERIALS AND METHODS
Materials
Peroxidase-conjugated sheep anti-mouse IgG, rhodamin-conjugated goat anti-mouse IgG and fluorescein-conjugated goat anti-rabbit Ig were purchased from Cappel (Turnhout, Belgium). A rabbit anti-p62 antiserum was produced previously (Saito et al., 1995). Leupeptin was from Peptide institute Inc. (Osaka, Japan).
Cell Culture and Preparation of Mitotic Cells
HeLa cells (RCB0007; just like ATCC CCL2, HeLa) and SP2/0-Ag14 myeloma cells (RCB0209) were obtained from Riken Cell Bank (Tsukuba, Japan) and were grown as described previously (Hosoya et al., 1997). HeLa cells grown to a subconfluent level on large culture dishes (245×245 mm; Sumitomo Bakelite Co., Tokyo, Japan) were treated with nocodazole at the final concentration of 0.25 μg/ml for 16 hr. Mitotic cells were detached by moving the media across the dishes with a pipette, collected by low-speed centrifugation and washed with PBS.
Production of monoclonal antibodies against the mitotic HeLa cells
Monoclonal antibodies were raised against the mitotic HeLa cells as described previously (Okubo et al., 1999). An 8-weeks-old female BALB/c mouse was immunized with 5×107 cells/ml of mitotic HeLa cells. After immunization, the spleen cells were fused with myeloma cells. Hybridoma cells were screened using the solid-phase ELISA with mitotic HeLa cells and 14 strains of them were selected for their high reactivities (over three times the absorbance of the control). Among these, one monoclonal antibody showed the dynamic changes of its staining pattern of HeLa cells throughout cell cycle and was designated as mH2. For immunofluorescence staining and immunoblotting, hybridoma culture supernatant was used. Production of ascities and purification of IgG fraction from the ascities were described previously (Okubo et al., 1999).
Indirect Immunofluorescence
Indirect immunofluorescence staining was performed as described previously (Okubo et al., 1999). Stained cells were examined with a Nikon Optifluorescence microscope. Photographs were taken with Fuji film, ISO400.
Preparation of HeLa cell extract
5×107–8 HeLa cells were harvested and mixed with F buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EGTA, 200 mM NaCl, 1 mM PMSF, 10 μg/ml leupeptin and homogenized on ice. The homogenate was centrifuged at 50,000 g for 30 min and the supernatant was collected.
Amino acid sequence analysis of mH2 antigen
The monoclonal antibody mH2 purified from mouse ascites was coupled to protein A Sepharose column (Ampure PA kit) according to manufacturer's instruction. After coupling, the column was washed with the binding buffer. The antigen-containing fractions were obtained by a gel filtration using Sephacryl S-300 column and applied to mH2 antibody-protein A Sepharose column. The column was washed with the binding buffer and then the mH2 antigen was eluted with the elution buffer (Ampure PA kit). To neutralize, 1 ml of 1 M Tris-HCl, pH 8.8 was added into the eluted fraction. The procedures for digestion of mH2 antigen with V8 protease and the determination of the amino acid sequence of the digested fragments were described previously (Okubo et al., 1999). Amino acid sequences were analyzed with an Applied Biosystems amino acid sequencer model 477A (Perkin Elmer Corp., Foster City, CA).
Other procedures
SDS-PAGE and immunoblotting were carried out in the same manner as described previously (Murata-Hori et al., 1998).
RESULTS AND DISCUSSION
Immunofluorescence observation using mH2 revealed that the mH2 antigen was localized at the nuclei in HeLa cells of interphase (Fig. 1, arrowheads) and diffusely in the cytoplasm of mitotic HeLa cells (Fig. 1, arrows). Immunoblot analysis of the cell extract with mH2 showed that it reacted with a single 86-kD band in both interphase and mitotic HeLa cell extracts (Fig. 2). To clarify the molecular aspects of the mH2 antigen, we determined the N-terminus amino acid sequence of the partial V8 protease digests of mH2 antigen (Fig. 3). We initially fractionated the HeLa cell extract using a gel filtration column, collected the fractions containing mH2 antigen and then purified mH2 antigen using protein A column coupled with mH2. The obtained mH2 antigen was digested with V8 protease and electrophoresed on SDS-PAGE. The transferred fragments onto PVDF membranes were subjected to amino acids sequencing. Each of four informative peptide sequences was highly homologous to that of an 86-kD subunit of human DNA helicase II (p86) (Fig. 3).
Fig. 1
Comparison of the subcellualr localization of mH2 antigen between interphase and mitotic HeLa cells. These panels are same fields. (a) Immunostaining with mH2. (b) DNA staining with 4′,6-diamidino-2-phenylindole (DAPI). (c) Nomarski differential-interference micrographs. Arrows indicate mitotic cells. Arrowheads show nuclei in interphase cells. Bar 5 μm.
Fig. 2
Immunoblot analysis of the mH2 antigen. Total cell extracts prepared from interphase (lane 1) and mitotic (lane 2) HeLa cells were separated on SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with mH2. A 86-kD band was detected in both interphase and mitotic cell extracts. Molecular weight markers are shown with molecular mass in kD (M).
Fig. 3
Comparison of partial amino acid sequences between the mH2 antigen and p86 subunit of HDH2. Partial amino acid sequences were determined on the four fragments generated by digesting the mH2 antigen with V8 protease. X represents undetermined amino acids. The starting and the ending amino acid position in p86 subunit of HDH2 are indicated at the left and the right, respectively. Amino acid identities are shown by boxes.
To analyze the specificity of mH2, HeLa cells in the various stages of the cell cycle were stained with mH2 (Fig. 4). p86 was localized at the nuclei in interphase cells (Fig. 4, a– c, arrowheads). When the cells entered into prophase, p86 was diffusely distributed throughout the cytoplasm except for chromosomes, mitotic spindles and constricted area of dividing cells (Fig. 4, d–f). And then, p86 was continuously diffused throughout the cytoplasm during mitotic phase (Fig. 4, g–r). Thus, the distribution of p86 dynamically changed from interphase to mitotic phase, but its localization was not changed during mitosis. These observations well coincided with those shown by Koike et al. who used the anti-Ku80 polyclonal antibody (1999).
Fig. 4
Subcellular localization of p86 subunit of HDH2 throughout the cell cycle in HeLa cells. The cells were interphase (a–c), prophase (d–f), metaphase (g–i), anaphase (j–l) and telophase (m–r), respectively. (a, d, g, j, m and p) Nomarski differential-interference micrographs. (b, e, h, k, n and q) DNA labeled with DAPI. (c, f, i, l, o and r) Immunofluorescence staining with mH2. The mH2 antigen was localized diffusely throughout the cytoplasm during mitosis.
Furthermore, we double-stained the interphase HeLa cells with mH2 and an antibody against GlcNAc-nucleoporin (p62) which was a component of nuclear pore complex and localized at the nuclear envelope (Davis and Blobel, 1986; Saito et al., 1995) (Fig. 5). Using the laser scanning confocal microscopy, we did not observe colocalization of p86 with p62 at the nuclear envelope. Also we did not observe that p86 was localized with any intracellular structures and nuclear periphery. This result corresponded to that of the biochemical aspect by Vishwanatha et al. (1995). For conclusion, a monoclonal antibody, which specifically recognizes an 86-kD subunit of human DNA helicase II, was produced.
Acknowledgments
We thank Y. Uji for helping the cell culture. This work was supported in part by grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to M. M.-H. and grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H. H..
