Monoclonal antibodies to mitotic cells.

Abstract

Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, we raised monoclonal antibodies to mitotic cells. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa cells were fused with SP2/0-Agl4 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine, and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species tested were isolated. Chromosomes as well as cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins from mitotic cells were separated by electrophoresis in NaDodSO4/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 0.40 to >200 kilodaltons (kDa). Both antibodies reacted strongly with three polypeptide bands of 182 kDa, 118 kDa, and 70 kDa. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were found to be phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.The nucleus of a mammalian cell undergoes profound reorganization when the cell enters mitosis. The nuclear envelope breaks down, and the chromatin condenses into discrete chromosomes. Although the initiation of mitosis is dependent on both RNA and protein synthesis until 2 hr and 1 hr, respectively, before mitosis, the addition of inhibitors of RNA and protein synthesis to cells after these times does not affect either initiation or completion of mitosis (1, 2).The fusion of a mitotic cell with an interphase cell can induce nuclear envelope breakdown and premature chromosome condensation of the interphase nucleus (3). During this process, [3H]tryptophan-labeled proteins from mitotic cells labeled at G2 phase become associated with the prematurely condensed chromosomes, whereas the grain density on the metaphase chromosomes is not changed (4). Chromosome condensation and germinal vesicle (nucleus) breakdown can be induced in frog oocytes by the injection of extracts from mitotic but not interphase cells (5). The activity is labile to heat, sensitive to protease, insensitive to nuclease, and stabilized by phosphatase inhibitors and is found both in the cytoplasm and on the chromosomes of mitotic cells (6). These results suggest that certain nonhistone proteins, possibly phosphoproteins, of mitotic cells are not present in interphase cells. Al-Bader et al. (7) have reported the presence of such mitotic-specific proteins by using two-dimensional gel electrophoresis.The objective of this study was to raise monoclonal antib...

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

“…To examine whether ECT2 is phosphorylated at Thr-Pro (TP) and/or Ser-Pro (SP) sequences in vivo, we immunoprecipitated endogenous ECT2 from interphase and mitotic cell extracts and subjected the immunoprecipitates to immunoblotting by two phosphospecific antibodies. The anti-pTP antibody was raised against the Cdk1 phosphorylation site in p21, and recognizes phospho-TP (pTP) (Thiel et al, 2002), whereas the anti-MPM2 antibody was raised against mitotic centrosomes, and recognizes both pTP and pSP motifs (Davis et al, 1983). ECT2 immunoprecipitates prepared from interphase or mitotic HeLa cell lysates were subjected to immunoblotting analyses with these antibodies (Figure 5a).…”

Threonine phosphorylation is associated with mitosis in HeLa cells

“…This protein was not one of the three major protein bands detectable in our earlier studies, probably because of the differences in extraction buffers used. In our initial studies [7], we extracted cells in phosphate-buffered saline (10 mM Na2HPO4/0.15 M NaCI; pH 7.3) (PBS), whereas here, we used 0.2 M NaCl buffer containing 10 mM Na2HPO4/NaHEPO4, 2 mM EGTA, and 10 mM MgSO4 supplemented with protease and phosphatase inhibitors [9]. Probably p55 was either not extractable with PBS or was dephosphorylated during extraction, since no phosphatase inhibitors were used in the earlier study.…”

“…The monoclonal antibody MPM-2 specifically stains cells undergoing mitosis and, on immunoblots, recognizes a family of phosphopeptides in the extracts of mitotic cells but not in those of interphase cells [7]. Of these phosphopeptides, a protein of approx.…”

“…A close correlation exists between the phosphorylation of certain proteins and the initiation of mitosis (and meiosis) on the one hand and the dephosphorylation of these proteins with the completion of mitosis on the other [4][5][6]. Although no currently available practical method blocks the phosphoryla-tion of these mitosis-specific proteins and thus the entry of cells into mitosis, it has been possible to use monoclonal antibodies (MPM-1 and MPM-2) that are specific to phosphoproteins in mitotic cells [7] to block the dephosphorylation of these phosphoproteins and thus prevent the transit from mitosis to the G1 phase [8]. When MPM-1 or MPM-2 was microinjected into interphase HeLa cells or two-cell stage frog embryos, the antibody did not inhibit the entry of cells into mitosis but did inhibit the exit of cells from mitosis [8].…”

“…These antibodies do not block mitosis because they recognize the proteins only after they are phosphorylated. Antibodies bound at or near the site of phosphorylation of the protein could inhibit its dephosphorylation, which is essential for the transition from mitosis to interphase [7,8]. The objective of our study was to isolate these mitosisspecific phosphoproteins and determine which of the three amino acids-serine, threonine and tyrosine -are phosphorylated and dephosphorylated during mitosis.…”