HYBRIDOMA Volume 9, Number 5, 1990 Mary Ann Leibert, Inc., Publishers
Cytotoxic Monoclonal Anti-Leukemia Antibody Binds to Histone HI JAMES M. SORACE and ROBERT J. JOHNSON
Baltimore DVA Medical Center and
Department of Pathology, University of Maryland School of
Medicine, Baltimore, MD
antibody (MAb) AP64 is a mouse IgM MAb raised against human non-lymphocytic leukemia (ANLL) cells. It has been shown to bind to a wide variety of cell lines and is capable of initiating complement (C) dependent cytotoxicity. Other studies indicated that MAb AP64 can effect long term cure in a leukemia minimal residual disease model. By using various techniques we have determined the identity of a protein which is bound by this MAb. Monoclonal
Immunofluorescent studies have shown that MAb AP64 stains the nuclei of fixed cells as well as metaphase chromosomes, indicating that this MAb binds to a Biochemical characterization revealed that MAb AP64 component of chromatin. western blots a 31 and 32 kilodalton doublet from NP-40 extracts from both rat and human leukemia cells. The mobility of this doublet is identical under reducing and non-reducing conditions. Further studies have shown that the bands detected by western blot analysis using MAb AP64 as a probe have a similar migration to those of bovine histone HI. Also, 1 nanogram of bovine histone HI can be detected by MAb AP64 when spotted onto nitrocellulose. These data demonstrate that MAb AP64 binds to a conserved epitope present on molecules coded for by the histone HI gene family. INTRODUCTION We have been studying mouse MAb AP64, which was raised against human ANLL Previous studies have shown that it binds to the surface of human and rat cells. ANLL cells. These studies have also shown that MAb AP64 can effect long term survival in a model of minimal residual disease in rats (1,2). We have been investigating the characteristics of molecule)s) which express epitopes recognized by this MAb. Although MAb AP64 is directed against cell membrane antigens, initial studies indicated that delipidated fixed cells bound more antibody than unfixed cells suggesting intracellular expression as well as cell membrane expression of the epitope. Immunofluorescence techniques were used to determine the intracellular distribution of the antigen(s) recognized by MAb Western blot and dot blot experiments were performed using a variety of AP64. protein preparations and peptide digests to study the antigen(s) involved in the nuclear binding of MAb AP64.
MATERIALS AND METHODS
Culture cell lines:
HeLa cells were grown on
supplemented with 10* FCS, 2 mM L-glutamine, 100 streptomycin/ml (MA. Bioproducts). HL60 ANLL cells culture under comparable conditions.
glass coverslips in RPMI 1640 penicillin/ml, and 100 ug were also grown in suspension
HeLa cells grown as above were fixed for 20 min in 70% Immunofluorescence: ethanol at 4° and then rinsed with PBS (.15 M NaCl, .015 M Na.HPO., pH 7.2). Next, they were incubated with a 1:100 dilution of ascites fluid containing MAb in PBS for 30 min at room temperature (RT) and rinsed in PBS three times. Fluorescein conjugated goat anti-mouse IgM (Southern Biotechnology) diluted 1:20 in PBS was then pipetted over the coverslip and incubated for an additional 20 min at RT. The coverslips were washed three times in PBS, mounted on slides using one drop of phosphate buffered glycerol, and sealed with nail polish. Additional experiments used a commercially available prefixed anti-nuclear antibody detection system purchased from Kallestad (Quanta Fluor Fluorescent Autoantibody Test using HEp-2 cell line substrate) and stained as above. In some experiments the slides were treated with .1 N HC1 according to the method of Tan et al. (3) Proteins: Purified histone HI was purchased from Boehringer Mannheim in lyophilized form and resuspended in distilled water to 2 mg/ml, aliquoted, and stored frozen as a stock solution. This preparation was observed to migrate as a doublet on SDS polyacrylamide gels stained with .1% Coomassie blue. Proteolytic enzyme digests were carried out according to the method of Böhm et al. (4). Briefly, Staphylococcus aureus V8-protease and chymotrypsin were purchased from the Sigma Chemical Company. Bovine histone HI was dissolved in 0.2 M ammoniumcarbonate buffer (pH 7.8) at a concentration of 2 mg/ml and incubated at 37° at an enzyme:substrate ratio of 1:200 for the indicated length of time. The reaction was terminated by addition of an equal volume of gel loading buffer, boiling for 5 min and immediate freezing. Rat ANLL cells were obtained by harvesting the spleens of hybrid (Lewis x Brown Norway) Fl rats 15-20 d after inoculation with 10 leukemia cells. The spleens were cut into small pieces and then forced through a wire mesh. These preparations contained 80-95% leukemia which were greater than 95% viable by dye exclusion. After harvesting, the cells were washed in PBS and treated with diisopropyl fluorophosphate (Sigma), .02ml Next the cells were extracted. cells for 30 min. Between 2-4 x 10 per 10 leukemia cells per ml were suspended in lysis buffer containing 20 mM Tris-HCl, 5 mM iodoacetamide and .5% NP-40 for 1 pH 7.4, 150 mM NaCl, 1 mM EDTA, .03% hr on ice with periodic agitation. The extracts were clarified by centrifugation at 3000 G for 5 min at 4 Human HL60 ANLL cells were extracted in a parallel fashion from cells harvested from tissue culture preparations. A 2M NaCl extract of calf thymus chromatin was the generous gift of E. N. Moudrianakis and was isolated from purified nuclei as previously described (5). Nitrocellulose squares, 1.5 cm across, were spotted with Dot Blot Assay: 2.5 ul of histone HI diluted in buffer containing 62.5 mM Tris, pH 6.8, 4M urea, 1 mM EDTA, 2% SDS and allowed to air dry. Next, the nitrocellulose was blocked for 20 min in PBS containing .5% Tween 20 (PBS-T) and then incubated overnight at 4° with a 1:11 final dilution of MAb containing tissue culture supernatant in PBS-T. The squares were washed 3 times in PBS-T and incubated with a 1:50 dilution of Iodine-125 labeled goat anti-mouse IgM antibody for 2 hrs at RT. Finally, the squares were washed as before and counted in a gamma counter. Data In some experiments the dot blots were is presented as the mean of triplicates. .
autoradiographed. SDS-Polvacrvlamide Gel Electrophoresis: The method of Laemmli was used with 10 or 12% slab gels and 4% stacking gels (6). Gels were run at RT overnight at 50 V. Next they were electroeluted onto nitrocellulose. Initial studies used a transfer buffer consisting of 25 mM Tris base, .82 M glycine, and 30% methanol. The purified histone HI preparations were found to transfer more efficiently if .01% SDS was added and methanol was reduced to 20%. Next, the nitrocellulose was probed with MAb as described above, and analyzed by autoradiography. Proteins in the gels were stained with Coomassie blue.
Antibodies: Production of the IgM MAb AP64, and control IgM MAb RM124, MH157, and RM152 have been described (1,7). Spent tissue culture supernatant was used in all dot blot and western blot experiments. MAb in the form of ascites fluid as well as tissue culture supernatants were used in immunofluorescence experiments with equivalent results. The amount of IgM MAb in the RM124 supernatant was comparable to that in the MAb AP64 supernatant when measured with iodinated goat anti-mouse IgM in a dot blot assay. RESULTS
Previous studies revealed that MAb AP64 binds to ANLL cells (1) and that One possibility was that MAb its binding was increased in delipidated cells. To further AP64 bound to epitopes expressed on intracellular antigen(s). evaluate this possibility, immunofluorescence studies were undertaken to Initial studies determine the intracellular distribution of this antigen(s). using HeLa cells revealed prominent nuclear fluorescence. A similar pattern of nuclear immunofluorescence was observed in ARL 16 cells (a rat liver cell line), and HL60 cells treated in the same fashion. Further studies were carried out using a commercially available preparation of the HEp-2 (human hepatoma) cell line used to detect anti-nuclear antibodies Again, prominent nuclear fluorescence was observed (Figure 1). No (ANA). nuclear staining was observed with the RM124 control antibody or with secondary Interestingly, numerous metaphase chromosomes were observed to antibody alone. This suggested that the antibody may be show binding by the MAb AP64. recognizing an epitope found on a molecule involved in chromatin structure. Acid extraction with .1 N HC1 totally abolished this fluorescence, suggesting that it bound to an acid extractable protein.
Localization of the Antigen Bound by MAb AP64 by Immunofluorescence. cell line was treated with MAb AP64 and then with fluorescein conjugated goat anti-mouse IgM. Note the cells show prominent nuclear staining in every nucleus in the field. Membrane or cytoplasmic fluorescence is rarely detected in the delipidated/fixed cells used in these experiments. Also, there is prominent staining of two metaphase plates in the upper right corner. FIGURE 1.
Western blot analysis of SDS-PAGE gels of rat leukemia and human HL60 extracts showed that MAb AP64 identified a species-conserved protein doublet that migrated at approximately 31 and 32 kilodaltons under reducing (Figure 2,) or non-reducing conditions (data not shown). Further studies using a high salt nuclear extract from purified bovine calf thymus nuclei revealed the detection of
Species Cross-reactivity of the MAb AP64. Extracts of either rat leukemia cells (left) or HL60 cells (right) were subjected to 10% SDS-PAGE and electroeluted onto nitrocellulose paper. The paper was cut into strips and incubated with spent tissue culture containing either MAb RM124 (A strips), MAb RM152 (B strips), MAb AP64 (C strips), MAb MH157 (D strips), or no MAb (E strips). Each of these MAb were of the IgM isotype. Following washing, bound MAb was detected by incubating Iodine-125 labeled goat anti-mouse IgM, washing, and performing autoradiography. FIGURE 2.
12% Antigenic Activity in High Salt Nuclear Extracts. Panel A polyacrylamide gel was loaded as follows: lane 1, 4 ul of a 2M NaCl nuclear extract; lane 2, 2ug of bovine histone HI and stained with Coomassie blue. Panel B loaded as Western blot of a gel run in parallel with the gel shown in A, and follows: lane 1, .4 ul of a 2M NaCl extract; lane 2, 2ug of bovine histone HI. HI migrates as a doublet just above the 29 kilodalton molecular weight standard and is detected by both western blot and protein staining. FIGURE 3.
doublet of similar mobility to the histone HI (Figure 3). Although having a calculated molecular weight of about 22 kilodaltons, histone HI from a variety of species is consistently reported to migrate as a doublet in the 30 to 34 These data, combined with the kilodalton range using SDS-PAGE methods. immunofluorescence results, suggested that the antibody recognized a conserved epitope coded by the histone HI gene family. To evaluate this, purified calf thymus histone HI was used in a dot blot assay (Figure 4). These results indicated that the MAb AP64 can detect less than 10 ng of histone HI. Additional dot blot experiments using autoradiography have detected approximately 1 ng of histone HI (Figure 4). To investigate where in the molecule the MAb AP64 binding epitope resides, bovine histone HI was digested using V-8 protease and chymotrypsin. The results V-8 protease cleaves at reproduced previously published banding patterns a
MAb AP64 Binds to Bovine Histone HI. FIGURE 4. Top: Various amounts of bovine histone HI were spotted onto nitrocellulose. MAb AP64 was allowed to bind and Results then detected with an Iodine-125 labeled goat anti-mouse IgM antibody. represent the mean of triplicates and the error bars indicate + 1 SD. Background was < 2500 CPM and was subtracted from all groups. Bottom: One nanogram (A and B) or 10 ng (C and D) of bovine histone HI were spotted in 10 ul of buffer onto nitrocellulose strips. The strips were then treated with MAb AP64 (A and C) or control MAb RM124 (B and D) followed by Iodine-125 labeled goat anti-mouse IgM. This autoradiograph represents a 36 hr exposure using image intensifying screens.
Panel A 12% polyacrylamide of Histone HI. follows: lane 1, 4 ug of histone HI; lane 2, a partial V-8 protease digest of 10 ug of histone HI; lane 3, a 5 min chymotrypsin digest of 10 ug of histone HI; lane 4, a 25 min chymotrypsin digest of 10 ug of histone HI. The gel was stained with Coomassie blue. Panel B Western blot of a gel run in parallel with the gel shown in panel A. Lanes 1 through 4 were loaded as in panel A. Note the positive bands are limited to the intact molecule or the fragments seen in lane 2. FIGURE 5.
glutamine, position 42, and presumably also at glutamine, position 52, resulting in a splitting of each band of the doublet (4). The V-8 protease treated fragments containing the hydrophobic and C-terminal domains of histone HI are detected by AP64 (Figure 5). Chymotrypsin, on the other hand, cleaves initially at a phenylalanine at position 104 and under prolonged incubation will further digest the molecule into multiple fragments (4,8). Interestingly, chymotrypsin digestion results in a complete loss of AP64 binding activity (Figure 5). These results suggest that the epitope resides near the phenylalanine at position 104. DISCUSSION We have found that MAb AP64, an MAb that mediates suppression of leukemia cells in vivo (1,2), recognizes an epitope on histone HI. Histones have been widely studied and much structural information is available. The histone HI gene family consists of a family of approximately six proteins (9, 10) which undergo a variety of post-translational modifications, including hormone-dependent phosphorylation (11). In addition, there have been reports of tissue specific With the electrophoretic expression of additional histone HI genes (12). resolution achieved in our studies, we observed that MAb AP64 bound to at least two of the isoforms of histone HI. At the protein level, histone HI molecules have three domains. The C-terminal and N-terminal domains are lysine rich, but The central domain is hydrophobic and highly conserved vary between species. The phenylalanine at amino acid position 104, which was found to among species. be involved in the MAb AP64 binding epitope, is in this conserved region. The amino acid sequence and structural information supports the conclusion drawn from the cross-species binding data, i.e. MAb AP64 binds to a species conserved epitope which is expressed on histone HI.
There have been reports concerning the detection of antigens on cell membranes that also are expressed in the nucleus, including histone HI (13-24). Cosgrove et al. have reported the identification of an MAb which reacted against histone HI as well as a 199 kilodalton protein (not histone HI) found on human platelets. This antibody also inhibited ristocetin induced or collagen induced platelet aggregation (13). Because of these findings, we tested human platelet extracts by western blot analysis using MAb AP64, but no bands were observed in this molecular weight range. Further, MAb AP64 has no inhibitory effect on
platelet aggregation (T. Kickler, personal communication). Thus, it is unlikely that MAb AP64 recognizes the same epitope as the MAb described by these authors. In another study. Holers and Kotzin observed that the IgM MAb MH-2 bound to MAb MH-2 binds to the cell membranes of human peripheral blood monocytes (14). histones H2A, H3, and H4, but not HI. These authors speculated that the binding of ANA may be involved in the pathogenesis of autoimmune diseases either by Our earlier studies concerning the immune modulation or by direct cytotoxicity. destruction of leukemia cells by MAb AP64 offer strong evidence that anti-histone antibody can cause direct cell killing in vivo (1,2). Other ANA, like MAb AP64, that bind to antigens expressed on the cell membrane could potentially have similar activity. Jacob et al. (15,16) and Kubota et al. (17) have studied the cell surface reactivity of anti-dsDNA MAb; MAb PME77 and MAb 2C10 respectively. In both cases, the MAb was also reactive with protease solubilized peptides released from viable cells migrating at approximately 34, 33, 17, 16, and 14 kilodaltons, designated as a lupus-associated membrane protein (LAMP). Kubota et ai. (17) have, in their system, provided evidence that the binding of MAb 2C10 to cell membranes is mediated by the bridging of the anti-dsDNA MAb and the cell surface histones by dsDNA. Our studies suggest direct binding of MAb AP64 to histone HI. The resolution of the nature of the target epitopes in these studies requires further investigation. Although the functional activities of histone HI are not completely understood, nuclear and membrane associated activities have been investigated. Histone HI is thought to play a significant role in chromatin condensation and gene regulation as well as to be an in vitro activator of a purified membranous protein kinase (25). To investigate the possibility that MAb AP64 mediates cellular cytotoxicity as a consequence of inhibition of the function of a vital cellular process, we studied the growth of HL60 leukemia cells in vitro. Neither the growth rate nor viability of the cells were effected by the addition of saturating concentrations of MAb AP64, as determined by radioimmunoassay, over a This finding suggests that MAb AP64 seven day period (unpublished observation). initiated immune mechanisms are responsible for cellular cytotoxicity rather than functional inhibition. In summary, our studies are unique in that they describe an MAb which is capable of initiating C dependent cellular cytotoxicity in vitro and suppression These results may be of leukemia in vivo (1,2) and also recognizes histone HI. important not only with respect to the mechanisms of immunotherapy of leukemia with MAb, but also with respect to understanding the mechanisms of autoimmune diseases as well. ACKNOWLEDGMENT We thank Dr. Scott Kaufmann for his suggestions and technical guidance, Deborah Howard-Shamblott and Marcey Shumway for expert technical assistance, Dr. Evangelos N. Moudrianakis for the bovine nuclear extract, Dr. Thomas Kickler for his assistance in the platelet aggregation studies, and Helen Spencer and Dr. Larry Brown for their preparation of the manuscript. REFERENCES
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Sorace Service (113) Baltimore DVA Medical Center 3900 Loch Raven Boulevard Baltimore, MD 21218
publication April 19,
Accepted after revision