Potu N. Rao

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells.

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8-10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation-dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.

Partial purification and characterization of mitotic factors from HeLa cells

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Inhibition of DNA polymerase α by gossypol

Abstract Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.

The Ultrastructural Organization of Prematurely Condensed Chromosomes

SUMMARY In an effort to understand the arrangement of the basic 30 nm chromatin fibre within metaphase chromosomes, changes in the organization of prematurely condensed chromosomes (PCC) were examined as a function of progression through the cell cycle. The structural features of PCC observed under the light microscope were compared with those obtained by scanning electron microscopy. PCC with varying levels of condensation were obtained by fusing mitotic HeLa cells with interphase cells synchronized at different times in the cell cycle. PCC from G1 cells are composed of rather tightly packed bundles of tortuous chromatin fibres. The density of fibre packing along the longitudinal axis of G1-phase PCC is lower and less uniform than that of metaphase chromosomes. Early G1 PCC exhibit gyres suggesting a despiralized chromonema. The condensed domains in G1 PCC appear to be organized as supercoiled loops; whereas fibre-sparse domains consist of longitudinal fibres running along the chromosome axis. As cells progressed towards S phase, a greater proportion of highly extended regions containing prominent longitudinal fibres became evident in the PCC. The pulverized appearance of S-phase PCC under the light microscope corresponded to the highly condensed, looping fibre domains separated by more extended segments containing longitudinal fibres that are visualized using the scanning electron microscope. Active sites of DNA synthesis are implicated to be localized within extended longitudinal fibres. Post-replicative chromosome maturation extends through the G2 period and appears to involve rearrangement of the extended longitudinal fibres into packed looping-fibre clusters, which then coalesce. These observations support the model for packing DNA into chromosomes proposed in 1980 by Mullinger & Johnson. Briefly, this model suggests that the chromonema of each metaphase chromatid contains regions composed of folded longitudinal chromatin fibres as well as looping fibres that emerge from the axis at distinct foci. The final level of chromatin packing in metaphase chromosomes is attained by spiralization of the chromonema.

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic “pulverized” appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis‐specific monoclonal antibody MPM‐2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM‐2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2‐mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM‐2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM‐2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P‐labeled S‐phase and M‐phase HeLa cell extracts after immunoprecipitation with anti‐p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2‐M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM‐2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis‐related events.

Early initiation of DNA synthesis in G1 phase HeLa cells following fusion with red cell ghosts loaded with S-phase cell extracts☆

A modification of polyethylene glycol-mediated cell fusion procedure has been described and standardized for red blood cell mediated microinjection of proteins into cells in suspension. Using this procedure, proteins are routinely introduced into 50% of the target cells. We have applied this microinjection procedure to introduce cytoplasmic extracts obtained from S-phase HeLa cells into HeLa cells synchronized in G1. This experiment resulted in an accelerated entry of the G1 cells into S phase as measured by the incorporation of [3H]thymidine. This technique may provide a means to identify and characterize the S-phase factors responsible for the induction of DNA synthesis.

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.

Role of Nonhistone Protein Phosphorylation in the Regulation of Mitosis in Mammalian Cells

The postsynthetic modification of proteins via reversible phosphorylation-dephosphorylation by phosphoprotein kinases and phosphatases has been reported to be an important mechanism in the regulation of numerous intracellular events (15,26,30). A strong correlation between the phosphorylation of histone H1 and (H3) and chromosome condensation and initiation of mitosis has been shown by several investigators (7,8,11,13,14,18,21,23). More recently, it was shown that histone H1 is also phosphorylated during premature chromosome condensation (8,22,27). However, many investigators believe that the superphosphorylation of histone H1 alone is not sufficient for chromosome condensation (10,20,22,27,38). Furthermore, chromosome decondensation could still occur even when dephosphorylation of histone H1 was blocked in mitotic cells (38). There is increasing evidence to suggest that phosphorylation of nonhistone proteins (NHP) may also be required for mitosis-related events. For example, phosphorylation of high mobility group (HMG) proteins has been suggested to be responsible for the shutting off of gene transcription during mitosis (12,28,34), increased phosphorylation of intermediate filament proteins like vimentin at mitosis (17,33), phosphorylation and dephosphorylation of laminar proteins have been implicated in the dissolution and reformation of the nuclear envelope (19,25).