Preliminary Characterization of a Monoclonal Antibody (AS-2) against Cell Cycle Related Proteins

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described. AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

The production of new Mabs directed against cell cycle dependent nuclear antigens of human normal and tumor cells may be useful to better understand nuclear structure and function [4,8]. These Mabs may also clearly be used in the field of quantitative pathology for clinical applications as, for examples, in cancer diagnosis and prognosis [9].
We have produced a series of Mabs immunizing mice with nuclear extracts obtained from the K562 cell line. FCM was used in coupling with measurement of DNA content for screening, in order to select immediately the Mabs associated to proteins that were clearly cell cycle dependent in their expression and/or stainability.
Our attention has been focused on a Mab that we preliminarily describe here (AS-2). As it will be seen, AS-2 showed a clear cell cycle dependance and recognized three proteins with different molecular weights and sub-cellular localization.
Cell harvest was done at the 3rd day after seeding to obtain a large fraction of proliferating cells. The cells, washed two times with phosphate buffer saline (PBS) Dulbecco's MEM, were resuspended in 2 ml solution A (solution A: NaCl 100 mM; sucrose 300 mM; piperazine-N-N -bis 2-ethanesulfonic acid (PIPES) 10 mM; MgCl 2 3 mM; pH 6.8) containing 0.5% Triton X 100 and incubated on ice for 30 min, to obtain cytoplasmic lysis. Washing was done once in PBS, followed by incubation in PBS containing both DNase I and RNase at 0.5 mg/ml at 4 • C for 30 min. Washing was done twice and nuclei concentration was adjusted to 50 × 10 6 nuclei/ml in Freund's adjuvant solution (Sigma, St. Louis, USA) to be used for mice immunization.

Mice immunization, cell fusion and FCM hybridoma screening
Immunization was performed with purified/sonicated K562 nuclei using females of Balb/C mice three months old. After five booster injections, mice were sacrificed and the spleens were removed. Hybridomas, derived from the fusion of spleen cells and P3 murine-myeloma cells, were obtained following standard procedures [10]. Supernatant of growing hybridoma clones were isolated and screened by FCM. The positive clones were subcloned by limiting dilution and the supernatants were again screened as previously.

Colcemid block/release
In G2/M-block/release experiments, colcemid at 0.1 µg/ml was added to the cell culture medium of proliferating cells. Cells were collected after 1.5 h of treatment (block) or were washed and added with fresh medium before being harvested 2 h after washing (release).

Flow cytometry
We used FCM to screen the hybridoma supernatants and for the determination of the Ig class. We also used multi-parametric FCM to characterize the cell-cycle dependent antigen expression. Nuclei were isolated (as previously detailed) using K562 cells harvested during exponential growth and diluted to 20 × 10 6 nuclei in 2 ml solution A. Fixation was with formaldehyde at final concentration of 1.85% for 10 min at room temperature. Nuclei suspensions were washed twice with PBS and divided into different tubes with approximately 3 × 10 6 nuclei for each tube. Nuclei were centrifuged and incubated for 1 h on ice with the supernatants in a total volume of 200 µl followed by three times washings in PBS * . Routine screening of supernatants was performed by FCM monoparametric analysis by using a secondary anti-Igmouse polyvalent FITC-conjugated antibody added at 1 : 100 dilution in PBS * . Ig class determination of positive clones was performed by monoparametric FCM. Briefly, secondary anti-mouse Ig-specific Mabs FITC-conjugated were tested. Fluorescence emission was evaluated in comparison with negative controls obtained using secondary unspecific anti mouse FITC-conj. antibodies.
When positivity of primary Mabs could be demonstrated, cell cycle distribution/dependance of Mabrelated antigens was investigated by means of biparametric FCM. Nuclei were resuspended in 1 ml PBS containing RNase 0.5 mg/ml and propidium iodide (PI) at 10 µg/ml for list-mode multivariate FCM analysis and sorting as previously described in details [11]. Briefly, we used a FACS 440 dual laser flow sorter system (Becton Dickinson, Mountain View, CA, USA) on line with an IBM-compatible 486 PC or a PDP11 to acquire 4 parameters in list-mode, i.e., red (PI) and green (FITC) fluorescences and forward and perpendicular scattering. Acquisition, storage and data analysis was either with the Phoenix flow system (San Diego, CA, USA) or with the Consort 40 system (Becton-Dickinson) which allowed flexible analysis with use of the "gating" property from only individual or 2-combined parameters.
Results were shown as "isometric contours", which display a three-dimensional distribution of values for any combination of two parameters versus the corresponding number of events given as isocell contour lines.

Extraction and MW determination of whole cells proteins
About 50 × 10 6 K562 cells at day 3rd of exponential growth were collected and washed one time in cold PBS. Cells were then suspended in distilled water with 0.1 µM phenylmethyl-sulfonyl fluoride (PMSF) (Sigma) and 1 mM dithiothreitol (DTT) (Sigma) for 10 min on ice. After hypotonic lysis, the cell suspension was sonicated repeatedly to complete cellular and nuclear lysis. The sample buffer (sodium dodecil sulfate (SDS) 4%; glycerol 20%; Tris(hydroxymethyl)-aminomethane (TRIS) 1 M 12% pH 6.8; bromophenole-blue 0.01%; 2-mercaptoethanol 0.24 M) was added at 1 : 1 to the cellular solution and boiled for 5 min. The solution was then used for SDS-PAGE on 8% acrylamide-containing gels following standard procedures [12]. The AS-2 supernatant was used at a dilution of 1 : 2 in PBS containing heat-inactivated non-immune horse serum (HS) 1%. The secondary antibody, an anti-mouse polyvalent peroxidase-conjugated antibody (Dako, Glostrup, Denmark) was used at the 1 : 400 dilution.

Extraction and MW determination of cytoplasmic and nuclear proteins
About 100 × 10 6 K562 cells at day 3rd of exponential growth were collected, divided in two equal samples, and resuspended in 1 ml solution A (see Section 2.1.1), containing 1% Triton X 100. After being maintained for 30 min on ice, and repeatedly pipetted to complete the disruption of the cytoplasms, the suspension was centrifuged at 11,200 g for 10 min. The supernatant, corresponding to the cytoplasmic fraction, and the pellet, corresponding to the nuclear fraction were separately collected. Furthermore, the nuclear suspension was then repeatedly sonicated on ice. The sample buffer was added at 1 : 1 to the cytoplasmic solution and to the nuclear one and boiled for 5 min. The method proceeded as described above for whole cells proteins.
In each case MW determination was assessed by means of molecular weight standards (Sigma) in the range of 29-205 kDa run in parallel with respect to K562 proteins.

Immunofluorescence microscopy
Human K562 erythroleukemia cells and nuclei were cytocentrifuged to glass slides following standard procedures. Human HeLa cells, instead, were directly grown on microscopic slides. Fixation was done for 10 min in PBS containing 1.85% formaldehyde, then washed with PBS and permeabilized in solution A (see Section 2.1.1) containing 0.5% Triton X 100 for 10 min at 4 • C. The slides were washed in PBS containing 0.5% BSA and 0.1% Triton X 100 and then incubated for 1 h at room temperature with BSA 5% in PBS. After the incubation with BSA, the cells were washed again and incubated with AS-2 antibody (supernatant diluted at 1 : 2 in PBS containing HS 1%) for 1 h on ice as described previously [13]. Cells were then washed carefully and incubated with a second FITC-conjugated anti-mouse IgM antibody at a dilution of 1 : 100. DNA staining was obtained with the use of 4 ,6-diamidino-2-phenilindole (DAPI) at 0.5 µg/ml. Pictures (1000×) were taken using a Leitz DMRB immunofluorescence microscope (Wetzlar, Germany).

Results
FCM screening identified a monoclonal antibody that we named AS-2. Secondary anti mouse IgG and IgM FITC-conjugated antibodies clearly showed that AS-2 was more reactive with anti IgM. Fluorescence emission associated with anti-IgG, in fact, overlapped to the negative control, while the intensity emission associated to IgM was about ten fold higher (Fig. 1).
Western blotting performed on proteins recognized by AS-2 in whole K562 cells showed three clearcut bands of reaction (Fig. 2) of approximate 200, 70 and 60 kDa molecular weights.
Cytoplasmic and nuclear extracts from K562 cells were obtained separately by means of detergent extractions. The 200 kDa molecular weight band was shown to be present only in the cytoplasmic extracts   A 200 kDa band in the cytoplasms and two bands of 70 and 60 kDa in the nuclei extracts were found as indicated by arrows. Reproducibility of these data was repeatedly tested. (Fig. 3(A)), while the two bands of 70 and 60 kDa were both found together in the nuclear extracts ( Fig. 3(B)). Figure 4 shows the relative amount (or, alternatively, the stainability) of the AS-2 recognized proteins in association with DNA content using both nuclei and cell suspensions. One may observe that AS-2 associated fluorescence values (in logarithmic scale with full scale corresponding to 3 decades) using nuclei (Fig. 4(B)) or cells (Fig. 4(F)) were more than 10 times higher than the values of the control nuclei or cells stained with a IgM isotype unspecific antibody (respectively, Fig. 4(A) and Fig. 4(E)).
The slight increase of IgM (FITC) fluorescence from 2C to 4C DNA values in the controls, was unspecifically related to cell/nuclei size increase. One may also observe that AS-2 fluorescence was largely spread for 2C (G1 phase) and 4C (G2 and M phases) DNA values while it was more constant in between 2C and 4C (S phase). Colcemid block-release experiments were performed to block cells in the M phase and later to observe the appearance in the 2C compartment of the early-G1 cell/nuclei. An increase of the "M" subpopulation is clearly visible in Fig. 4(C) after colcemid block. At the same time, a subpopulation of cells with 2C DNA content and low/negative AS-2 fluorescence values completely disappeared. Colcemid release experiments (Fig. 4(D)) showed that this nuclei subpopulation (identified in Fig. 4(D) as early-G1) reappeared. Figure 4(D) also illustrates the dynamics of cells at the colcemid block to release transition in which an accumulation of cells in late-S and G2 phases with absence of late-G1 and early-S cells is clearly visible.
Sorting of cell/nuclei from the interphase and M compartments was done to help investigating the staining characteristics of AS-2. Nuclei from the early-G1 compartment showed very low to null AS-2 fluorescence. Nuclei at late-G1, S and G2 had a similar positive staining over the entire nuclei. Examples of these staining patterns are shown in Fig. 5. Figure 5 shows the staining characteristics of AS-2 in interphase and mitotic nuclei and in whole cells by immunofluorescence microscopy. DAPI staining of nuclei cytocentrifuged on slides after AS-2 immunostaining, allowed the identification of nuclei from interphasic cells (Fig. 5(A)) and mitotic events (window in Fig. 5(A)). The same fields in green fluorescence (i.e., AS-2 staining) showed that while two interphasic nuclei were positive, with a staining pattern revealing a regularly interdispersed fluorescence, one nucleus, probably belonging to early-G1 phase, were completely negative to AS-2 immunostaining (Fig. 5(B)). Mitotic events presented a positivity limited to two little, brilliant spots, opposite to each other that likely correspond to the cell centrosomes (window in Fig. 5(B)).

Discussion
A monoclonal antibody (AS-2) was obtained using K562 extracted nuclei as immunogens. AS-2 (IgM class) recognized three different polypeptides which differed in molecular weights (200, 70 and 60 kDa) and were located in the cytoplasm (200 kDa) and in the nucleus (70 and 60 kDa). The cytoplasmic proteins appeared easily removable, while the 70 and 60 kDa forms appeared to be strongly bound to nuclear structures.
Multiparameter flow cytometry was used to evaluate the amount of AS-2 positive proteins and DNA content. The highest AS-2 associated fluorescence was for cells or nuclei in the late-G1, S and G2 phases of the cell cycle. The lowest AS-2 fluorescence (at control level) was for early-G1 nuclei and mitoses. Evidence for these last observations was obtained using colcemid treatment that allowed to block cells in M and then to release the block and to observe cells entering in the early-G1 phase of the cell cycle after telophase.
AS-2 staining pattern has been investigated in both nuclei and cells. AS-2 fluorescence was mainly concentrated in two spots in M, disappeared almost entirely in early-G1 and was again evident over the entire nuclei in late-G1, S and G2 phases of the cell cycle. The proteins recognized by the AS-2 monoclonal antibody appear, therefore, to be degraded before the cells divide or keep a small concentration during the early-G1 postmitotic period. Moreover, these proteins appear to be synthesized up again during the late-G phase of the cell cycle and maintained at high concentration in S and G2 phases.
The present knowledge of the protein recognized by the AS-2 monoclonals is preliminary. The AS-2 recognized proteins in interphase appear to be associated with the nuclear matrix while in mitosis they are visible in two little centrosome-like spots. Colocalization experiments with known proteins of the nuclear matrix and of the centriole associated cell centrosomes are needed to deepen and complete these early findings. In the present study, AS-2 immunofluorescence coupled with FCM indicates a practical mean to separate the G1 phase of the cell cycle in two subphases, i.e., early-G1 and late-G1, with respectively absence and presence of the protein within the nuclei. Moreover, the FCM method we have used allows to clear-cut separate G2-phase cells from the M-phase cells.
AS-2 staining was studied on HeLa cells grown on slides and K562 cells grown in suspension. AS-2 staining was homogeneously dispersed over the entire cytoplasm. The relationship between the nuclear AS-2 recognized nuclear proteins and the AS-2 cytoplasmic proteins, beside the different molecular weights, is so far not known.
In conclusion, we have produced and preliminarily characterized a monoclonal antibody that appears to provide means to investigate the cell cycle in details and could be potentially useful in a number of clinical investigations based on mitoses counting. Further work needs to be done to better understand the role of the AS-2 associated proteins during the cell cycle progression.