Rose K. Busch

Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions

Abstract After extraction of Novikoff hepatoma nucleoli with 4 M urea/3 M LiCl, phosphoprotein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography. Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody as determined by Ouchterlony diffusion analysis. When the immunoperoxidase method was used to localize protein C23 in cells, it was found in ‘fibrillar centers’ (nucleolonemas) in nucleoli. Protein C23 was also demonstrated to be present on the nucleolus organizer regions (NORs) of metaphase chromosomes.

CENP-G: A NEW CENTROMERIC PROTEIN THAT IS ASSOCIATED WITH THE ALPHA -1 SATELLITE DNA SUBFAMILY

Abstract. A new constitutive centromere-specific protein (CENP) has been identified as a result of its recognition as an autoantigen by serum from a patient with gastric antral vascular ectasia disease. Conventional immunoblotting and two-dimensional double blotting with both this antiserum and a known anti-centromere antiserum showed that this antiserum predominantly recognized a Mr 95,000 protein that is different from all known CENPs. We have named this new protein CENP-G. This protein was detected at the centromeric region throughout the cell cycle. In mitosis, it was restricted to the kinetochore inner plate as shown by immunogold labeling and electron microscopy. The centromeres of some human chromosomes are known to contain two subfamilies of α-satellite DNA. Using immunofluorescence combined with fluorescent in situ hybridization with subfamily-specific DNA probes, we revealed that CENP-G was specifically associated with one of the subfamilies, which we have named α-1, but not the other. The localization and the α-1-specific association suggested that CENP-G may play a role in kinetochore organization and function. Like CENP-B and C, but unlike CENP-A, this protein remained with the nuclear matrix after intensive extraction. While CENP-B is absent from the human Y chromosome, the existence of CENP-G on the Y chromosome has been proven by immunofluorescence and whole chromosome painting. CENP-G was also detected in CHO, Indian muntjac and Chinese muntjac cells, suggesting that it is conserved in evolution.

Antigenic proteins of nucleolar chromatin of Novikoff hepatoma ascites cells.

Nucleolar chromatin of Novikoff hepatoma ascites cells contains an antigen (no-Ag1) detected with antinucleolar antibodies by the immunodiffusion technique. This antigen was distinguished from the previously reported nuclear chromatin antigen NAg-1 (19) by the findings that tumor nucleolar antibodies which formed immunoprecipitin bands with no-Ag1 did not do so with NAg-1 and that tumor cytosol, which contains NAg-1, formed immunoprecipitin bands with tumor chromatin antibodies but not with antibodies to tumor nucleoli. Tumor nucleolar chromatin contains both NAg-1 and no-Ag1, but only no-Ag1 formed bands with tumor nucleolar antibodies. no-Ag1 is a component of tumor nucleolar chromatin that was not soluble in 0.075 M NaCl - 0.025 M EDTA, pH 8, and only slightly soluble in 0.01 M Tris-HCl, pH 8. no-Ag1 was not found in liver nucleoli. Antibodies to liver nucleoli formed immunoprecipitin bands with liver nucleolar antigens but none were confluent with those formed between tumor nucleolar antibodies and antigens of tumor nucleolar chromatin. Absorption of the tumor nucleolar antibodies with whole tumor cells or whole liver pressate did not alter band formation with no-Ag1. Three antigens in liver nucleoli were not found in tumor nucleoli.

Immunodominant RNA recognition motifs of human nucleolin/C23

Nucleolin/C23 is a nucleolar phosphoprotein implicated in the synthesis, processing and transport of ribosomal RNA and gene transcription. Auto-antibodies to human nucleolin/C23 have been reported in patients with systemic lupus erythematosus and other systemic autoimmune disorders. To identify immunodominant regions in nucleolin/C23, deletion fragments of nucleolin/C23 were fused in frame with the glutathione S-transferase gene. Seven monoclonal anti-nucleolin/C23 antibodies were used to determine the immunoreactivity of the bacterially expressed fusion proteins. Two sets of immunogenic regions at amino acids 314-389 and 387-461 were identified; each contained overlapping discontinuous epitopes and a centrally located RNA recognition motif. An auto-immune serum from a patient with systemic lupus erythematosus patient was found to contain antibodies against human nucleolin/C23 which recognized amino acids 387-461 of nucleolin/C23.

An enzyme immunoassay for the detection of human tumor nucleolar antigens

An enzyme immunoassay (EIA) was developed in which nuclear extracts were bound to the wells of polystyrene microtiter plates. The presence of various antigens in the extracts could be detected using antiserum raised against HeLa cell nucleoli. Normal human cell extracts, coupled to Sepharose as a solid phase absorbent, were used to remove antibodies directed against antigens present in normal cells, The resulting purified antibodies produced a linear EIA absorbance in the range of 100-500 ng tumor extract, but little EIA absorbance with nuclear extracts from human placenta and liver.

Actinomycin D inhibition of monoclonal antibody binding to nucleolar phosphoprotein 37/5.2 (B23)

Actinomycin D was found to block binding of a monoclonal antibody to nucleolar phosphoprotein 37/5.2 (B23) when incubated either simultaneously with or prior to addition of the antibody. Daunorubicin had no significant blocking activity of this antigen-antibody reaction. In comparative studies with a monoclonal antibody to nucleolar phosphoprotein 110/5.2 (C23), actinomycin D exhibited little blocking activity. When a 42-mer peptide containing the epitope of protein 37/5.2 (B23) was tested as the antigen, similar inhibition by actinomycin D of binding of the monoclonal antibody was found. These results provide evidence for binding of actinomycin D to a specific epitope of protein 37/5.2 (B23).

Isolation and partial characterization of a nuclear antigen (68/6.3) from the Namalwa cell line (a Burkitt lymphoma)

SummaryA nuclear antigen was purified from the 0.01 M Tris-HCl/pH 8 extract of nuclei of the Burkitt tumor Namalwa cell line to electrophoretic homogeneity by DEAE cellulose chromatography, affinity chromatography, and preparative isoelectric focusing. The yield of antigens was 0.02% of the nuclear 0.01 M Tris-HCl/pH 8 extract. On two-dimensional gel electrophoresis, the major antigen separated into two adjacent protein spots with molecular weights of 68,000 and an approximate pI of 6.3 (68/6.3 A and 68/6.3 B). A minor antigen had a molecular weight of 61,000 and pI of 6.0 68/6.0). Fourteen 125I-labeled peptides were obtained from the tryptic digest of the major antigen (68/6.3 A and 68/6.3 B). The amino-acid composition analysis of the purified antigens indicated that the amino acids in the highest content were glycine (15%), glutamic acid (11.6%), and serine (9%); the ratio of acidic to basic amino acids was 1.95. In studies on nucleolytic activity, the purified antigen produced a single-stranded and then a double-stranded cleavage of PM 2 and pBR 322 DNA. This antigen is the first purified nuclear antigen that reacts with the HeLa-specific nucleolar antibodies.

Results of a preliminary "blind" study on the presence of the human tumor nucleolar antigen in breast carcinomas, benign breast tumors, and normal breast tissues.

Abstract A study was made on the presence in benign and malignant breast tumors of a common nucleolar antigen previously found in a broad range of human malignant tumors (Busch et al. , Cancer Res. 39 , 3024, 1979). The specimens were treated first with rabbit antibodies to “nucleolar preparations” or the nuclear 0.01 M Tris-HCl extracts of HeLa S3 cells and then with fluorescein-conjugated goat anti-rabbit antibodies. With this procedure, bright nucleolar immunofluorescence was observed in 19 20 (95%) of known breast cancer specimens. In the group of 80 unknown samples in the “blind” study, 75 (94%) were correctly identified as malignant or benign on the basis of the presence and distribution of the nucleolar fluorescence. In a group of 67 samples in which the nucleolar fluorescence was either readily observed or virtually absent, 47 48 (98%) of the malignant tumors were correctly identified. Of the benign lesions or normal breast specimens, 18 19 (95%) were correctly identified as negative for nucleolar fluorescence. These studies extend the results previously reported of a common nucleolar antigen in a broad range of human cancers to a larger series of malignancies of a particular organ. The tumor nucleolar antigen(s) were partially characterized by isoelectric focusing on 4% polyacrylamide gels. One major band had a p I of 6.3 and a minor band had a p I of 6.1. These antigens were not found in the normal human liver nucleoli.

Nucleolar immunofluorescence in human hematological malignancies

Abstract Rabbit antibodies to the nuclear Tris extract of HeLa cells which have been shown by the indirect immunofluorescence technique to localize in nucleoli of a variety of human malignant tumors but not in a number of nontumor tissues also produced bright fluorescence in nucleoli of tumor cells in several hematological malignancies. The tumors studied included Hodgkins malignant lymphoma, non-Hodgkins malignant lymphoma, acute myeloid and acute myelomonocytic leukemia, chronic lymphatic and chronic myeloid leukemia. In contrast, none of the corresponding normal cell lines in the bone marrow exhibited bright nucleolar fluorescence. In addition, neither the cells of patients with acute infectious mononucleosis nor lymphoid hyperplasia exhibited bright nucleolar fluorescence. These studies suggest that antibodies to HeLa cell nucleolar antigens may be useful in immunodiagnosis of human malignancies.

The effect of antisense p120 construct on p120 expression and cell proliferation in human breast cancer MCF-7 cells

Malignant transformation of NIH3T3 cells was observed by transfection with the pSVX vector containing a sense human p120 cDNA construct (pSVX120). Subsequent transfection of these transformed cells with a dexamethasone inducible antisense p120 construct (pMSG021) markedly reduced the expression of human p120 and the growth rate of these transformed cells (Perklaky et al., Cancer Res., (1992) 52, 428-436). In the present study, a human breast cancer cell line (MCF-7) which expresses the p120 protein was transfected by electroporation with a pSVX plasmid-construct containing the antisense p120 cDNA (pSVX021). Clones containing the pSVX021 construct were selected and analyzed for expression of p120 mRNA, protein and growth characteristics. The expression of the p120 protein was inhibited by 44% in the antisense-transfected MCF-7pSVX021 cells; a 56% inhibition of cell-growth and a reduced colony formation in soft agarose were also observed. The growth of MCF-7 cells transfected with the p120 antisense construct was reduced by 93% in nude mice.