Isolation of a 70 000 molecular weight antigen of the Novikoff hepatoma.

Previous reports from this laboratory have indicated that a number of cytosol and nuclear proteins of Novikoff hepatoma cells were immunologically related [Yeoman, L. C., Jordan, J. J., Busch, R. K., Taylor, C. W., Savage, H., & Busch, H. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3258; Busch, R. K., & Busch, H. (1977) Tumori 63, 347]. In preparation for analysis of their structure and function, studies were undertaken to purify nuclear antigen 2 from the cytosol of Novikoff hepatoma cells in high yield and purity. It was shown on Ouchterlony gels that cytosol nuclear antigen 2 formed a single immunoprecipitin band of identity with one of the bands extracted from Novikoff nuclear chromatin. In this study, a 70 000 molecular weight antigen was isolated from the cytosol of Novikoff hepatoma cells by ammonium sulfate fractionation, ion-exchange chromatography, and isoelectric focusing in a granulated gel bed. This protein which focused at a pI of 6.3 was labeled with 125I-labeled Bolton-Hunter reagent and purified on an Ultrogel AcA-44 column. As shown by electrophoresis on NaDodSO4-polyacrylamide gels, the antigen in the excluded volume migrated as a single protein with a molecular weight of 70 000. The overall purification over the starting material was 2890-fold.     

Nucleolar immunofluorescence in human hematological malignancies.

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.     

Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells

Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed.     

C-reactive protein reacts with the U1 small nuclear ribonucleoprotein

C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.     

Studies on the human tumor nucleolar antigens

With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat antirabbit antibodies, bright nucleolar immunofluorescence was observed in human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in most normal tissue specimens, benign adenomas, hyperplastic tissues, and specimens of inflammatory diseases. 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. 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 bening lesions or normal breast specimens, 18/19 (95%) were correctly identified as negative for nucleolar fluorescence. These studies extend the results previously reported for 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 pI of 6.3 and a minor band had a pI of 6.1. These antigens were not found in the normal human liver nucleoli.     

Detection of a 140 KDA nucleolar protein with an anti-PCNA autoantibody

To assess the numbers and types of PCNA (proliferating cell nuclear antigen) species, immunoprecipitation studies on HeLa cell, nuclear and nucleolar extracts were performed. A 140 KDa protein from HeLa nucleoli was immunoprecipitated by an autoantibody (E.B.) previously used to detect the proliferating cell nuclear antigens (PCNA). The 140 KDa protein was also detected in the nuclear extract of colon carcinoma cells (omega) labeled in vitro with 125I-Bolton Hunter reagent. When the growth of the colon carcinoma cells was inhibited by 1% N,N-dimethylformamide for two weeks, the 140 KDa protein was not detected which suggests this protein is associated with cell growth.     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

A Comparative Nuclear Localization Study of Galectin-1 with Other Splicing Components

Using both conventional and laser confocal fluorescence microscopy, the intracellular distribution of galectin-1 in HeLa cells was analyzed and compared with the localization of previously documented markers of the nucleus and cytoplasm. The Sm epitopes of the small nuclear ribonucleoprotein complexes (snRNPs) and the non-snRNP splicing factor SC35 yielded only nuclear staining. On the other hand, the enzyme lactate dehydrogenase was cytoplasmic. In contrast to these patterns in which nuclear versus cytoplasmic localizations appeared to be mutually exclusive, galectin-1, as well as galectin-3, yielded simultaneous nuclear and cytoplasmic staining. Confocal microscopy showed galectin-1 fluorescence throughout most of the sections from the top of the cell to the bottom. Through the middle sections, as the plane of focus cuts through the nucleus, there was definite fluorescence staining in the nuclear compartment. This nuclear localization was critically dependent on the type of detergent used to permeabilize the cell: cells treated with saponin or digitonin yielded exclusively cytoplasmic staining while Triton X-100-treated cells showed nuclear as well as cytoplasmic labeling. Finally, double-immunofluorescence analysis showed that, within the nucleoplasm, the following pairs of nuclear antigens could be colocalized in certain speckled structures: (a) SC35 versus Sm; (b) galectin-1 versus Sm; (c) galectin-3 versus Sm; and (d) galectin-1 versus galectin-3. These results establish the presence of galectin-1 in the nuclei of HeLa cells, a conclusion consistent with the identification of the protein in nuclear extracts of the same cells and with its documentation as a factor in pre-mRNA splicing.     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.     

Intracellular localization and tissue specificity of human uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) purified by various procedures from the human placenta was used to obtain immune antisera with specific antibodies, the antibodies being affinity-purified on UDG-sepharose. Two immunoreactive polypeptides were found in crude extracts of the human placenta with the help of the antibodies. Their apparent molecular masses were about 37,000 and 34,000 dalton. Only the former polypeptide was found in crude extracts of the human embryonal heart, liver and in HeLa cells. The indirect immunofluorescent staining shows both slight and intensive fluorescence of HeLa cell nuclei. The similarity of antigenic properties of the human and rat UDG was confirmed.     

Members of a family of proteins (the RD family) detected by a U1 70K monoclonal antibody are present in spliceosomal complexes.

We have characterized a monoclonal antibody (mAb) to the U1 snRNP component U1 70K. We find that this antibody recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K was previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus we have designated the novel mAb U1 70K antigens the RD family. Comparison of mAb U1 70K with a recently characterized antibody, mAb 16H3, whose epitope is a repeating R/D or R/E motif, showed that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors.     

Widespread occurrence of polypeptides related to neurotubule-associated proteins (MAP-1 and MAP-2) in non-neuronal cells and tissues.

The occurrence and cellular localization of polypeptides related to hog brain microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2) in non-neuronal cell lines of various species and types, and in several tissues from rat was studied. When insoluble cell fractions were prepared by incubation of isotonic cell extracts with 20 microM taxol, polypeptides co-migrating with MAP-1 and MAP-2 upon gel electrophoresis were observed in virtually all cases examined. Immunoblotting of preparations from 3T6, CHO, HeLa and N2A cells, as well as pituitary, heart, testis and liver revealed immuno-reactivity with antibodies to neuronal MAP-1 for polypeptides co-migrating with MAP-1 in all cases, except for HeLa cells and liver. With similar preparations, antibodies raised to neuronal MAP-2 were barely reactive with bands of the MAP-2 size except for N2A cells and pituitary gland. In all cases of non-neuronal cells and tissues, major cross-reactive bands, however, were of mol. wt. lower than that of MAP-2, indicating, most likely, proteolytic breakdown of MAP-2 during cell fractionation. As shown by double immunofluorescence microscopy of various cultured cell lines using affinity-purified antibodies to MAPs, and monoclonal antibodies to tubulin, MAP-1-as well as MAP-2-related antigens were generally, but not exclusively, associated with typical microtubule structures of the cytoplasm, spindle, midbody and primary cilia. Antigens related to both MAPs were also localized in frozen sections of rat trachea, testis, pituitary, kidney and cardiac and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.