Immunocytometric determination of the alpha and beta isoforms of human DNA topoisomerase II: influence of different fixatives.

The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The extent of antigen preservation achieved with the dehydrating fixatives, ethanol and acetone, was compared with that obtained with the cross-linking agent paraformaldehyde. Binding of each MoAb to the relative isozyme was detected by indirect immunofluorescence labelling and quantified by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated with specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isoform was observed after ethanol, and to a minor extent after paraformaldehyde fixation, while reactivity of the 170-kDa isoform was not significantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs. DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pattern of immunofluorescence showed that the localization of each isozyme was very similar for all these fixatives. These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the relative amount of topo II isoforms with immunocytometric techniques.

Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines.

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.

Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.

The sensitivity to DNA topoisomerase inhibitors in L5178Y lymphoma strains is not related to a primary defect of DNA topoisomerases.

DNA topoisomerase-targeting antitumor drugs are potent inducers of protein-concealed strand breaks in mammalian cells and act by trapping DNA topoisomerases on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes. It has been proposed that the cleavable complex is an unusual form of DNA damage that elicits cellular responses analogous to those caused by DNA damaging agents. The relationship between topoisomerase-targeting drug-induced damage and radiation-induced damage has been investigated by analyzing the properties of DNA topoisomerases in mouse L5178Y lymphoma strains that are cross-sensitive to topoisomerase I-II inhibitors and to UV light or X-ray irradiation. The strains are LY-R, isolated from L5178Y cells on the basis of increased resistance to ionizing radiation, and strain LY-S, isolated from LY-R cells following a spontaneous increase in the sensitivity to ionizing radiation. LY-S cells, deficient in the rejoining of DNA double-strand breaks, show enhanced sensitivity to topoisomerase II-targeting inhibitors, whereas LY-R cells have an increased sensitivity to UV radiation and to the topoisomerase I inhibitor, camptothecin. The cellular availability of DNA topoisomerase I and II and the sensitivity of the enzymes to their specific inhibitors have been measured in the two related strains. In the LY-R strain, we found a 30% decrease in topoisomerase I content but no difference in camptothecin sensitivity, while no quantitative or qualitative differences were observed for the topoisomerase II. The results indicate that variations in sensitivity of the L5178Y strains to topoisomerase inhibitors are unlikely to be related to primary defects of the target enzymes, and thus it is possible that common pathways exist for processing of topoisomerase- and radiation-induced damage.

Antibodies to topoisomerase II in idiopathic pulmonary fibrosis.

Antibodies to DNA topoisomerase II (anti-topoisomerase II) were detected by ELISA in the sera of 18 out of 41 (44%) patients with idiopathic pulmonary fibrosis (IPF). Follow-up sera were also obtained from 19 of the patients. DNA topoisomerase II binding remained constantly high or low in the majority of follow-up sera, but 2 out of the 8 positive cases became negative while 3 out of the 11 negative cases became positive during follow-up. No association was found between occurrence of anti-topoisomerase II antibodies and any indices of disease severity. Furthermore, individual patient follow-up did not show any correlation between changes in topoisomerase II binding and deterioration or improvement of clinical status. In conclusion our study shows that although anti-topoisomerase II are detectable in a large fraction (approximately 50%) of IPF patients and are useful for diagnostic purposes, they do not provide a measure of clinical activity.

DNA topoisomerase II beta: stability and distribution in different animal cells in comparison to DNA topoisomerase I and II alpha.

DNA topoisomerase I and the isoforms alpha and beta of DNA topoisomerase II were analyzed in different animal cells using a panel of monoclonal antibodies (MoAbs). The beta isoform is a most unstable enzyme. We investigated conditions to stabilize beta isoform because its variability changes according to the derivation of cells. We describe two MoAbs specific to DNA topoisomerase I: the first one recognizes the enzyme in all the species tested including fish; the second one, in contrast, recognizes an epitope present only in mammalian cells. We also found that eight of eight MoAbs against DNA topoisomerase II alpha and five of six against the beta isoform recognize the respective enzymes in all the species tested excluding fish. In addition, MoAbs to the alpha isoform are specific to epitopes not present in the carboxyl third of the enzyme.

Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by photosensitivity, a high incidence of cancer in sun-exposed portions of the skin and a reduced capacity to repair the u.v.-induced DNA damage. One of the XP mutations (XP-D) has also been identified in patients affected by trichothiodystrophy (TTD), a rare autosomal recessive disease characterized by brittle hair, mental and physical retardation, peculiar face and ichthyosis. However, in these patients there is no evidence of increased skin tumour incidence. Since an impairment of cell-mediated immunity has been proposed as a co-factor in the cancer proneness of XP patients, we investigated the involvement of immune defect(s) in five XP patients, five TTD patients, their parents, and 24 TTD relatives. We evaluated the phenotype of circulating lymphocytes, natural killer (NK) cell lytic activity, target cell binding of NK cells at single cell level and the effect of interferons (IFN) alpha and beta on NK cell activity. The relative proportion of CD3+ and CD4+ circulating lymphocytes was reduced in XP but not in TTD patients. NK cell lytic activity was decreased in XP patients and their mothers, but their fathers showed normal lytic activity. NK activity varied among TTD families: four out of five patients and their relatives presented low NK cell activity, and one family was normal. In TTD family members, NK activity increased after incubation with IFN-alpha or IFN-beta, but never reached normal values. In contrast, in XP patients and their mothers, the defect was almost completely corrected after in vitro incubation with IFN-alpha or IFN-beta. Our study indicates impaired NK lytic activity in the majority of TTD and XP patients and that this defect is present also in members of their families. In addition, XP patients present a low number of circulating T cells. These multiple abnormalities, together with DNA repair defects, could be related to the increased cancer risk in XP patients.

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes.

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.

The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant.

Monoclonal antibodies raised against two isoforms (170 and 150/180 kDa) of DNA topoisomerase II showed distinct fluorescence patterns in HeLa cells in different moments of the cell cycle (C. Negri et al., 1992, Exp. Cell Res. 200, 452-459). The ultrastructural distribution of the 150/180-kDa isoform, which in immunofluorescence showed a localization into the nucleolar region, has been analyzed by electron microscopy with a gold-conjugated secondary antibody in HeLa and K562 cells. The results indicate that this isoform of the enzyme is exclusively localized in the nucleolus, mainly in the dense fibrillar component, while the nucleoplasm of interphase cells and the chromosomes of mitotic cells are completely negative. The antibody also reacts with the nucleolus of isolated nuclei and with the nucleolar remnant of purified nuclear matrices. A quantitative evaluation of the label distribution indicates that the percentage of label in the nucleolar remnant of isolated matrix is almost identical to that of the nucleolus in whole cells. The interaction with the insoluble proteins of the isolated nuclear matrix is also demonstrated by quantitative immunoblotting in which the MoAb specifically stains a unique band corresponding to the 150/180-kDa isoform of topoisomerase II. The localization of the 150/180-kDa isoform of topoisomerase II in the nucleolar remnant strongly suggests that it represents a structural element for the spatial organization and for the regulation of transcription of the ribosomal genes.

Antibodies to hn-RNP protein A1 in systemic lupus erythematosus: clinical association with Raynaud's phenomenon and esophageal dysmotility.

Antibodies to recombinant hn-RNP protein A1 were found by ELISA in sera from 26 out of 67 unselected patients with systemic lupus erythematosus. A higher number of anti-A1 positive patients had Raynaud's phenomenon (50% vs 7%) and esophageal dysmotility (42% vs 5%) than the anti-A1 negative patients. All 8 patients with both Raynaud's phenomenon and esophageal dysmotility had a positive anti-A1 assay. No association was found with other clinical findings, nor with disease activity and treatment regimes. Anti-A1 antibodies did not correlate with anti-RNP and anti-Sm antibodies, which were present in 30% and 12% of the anti-A1 positive cases and in 22% and 7% of the anti-A1 negative cases, respectively. Our results indicate that antibodies to hn-RNP protein A1 may be associated with a subset of SLE patients with clinical features overlapping those of progressive systemic sclerosis and quite distinct from the group identified by anti-RNP antibodies.

Synthetic and natural factors promoting production of murine and human monoclonal antibodies.

Human endothelial culture supernatant (HECS) contains factor(s) which promote the growth and the stability of hybridoma cells, and in these respects is superior to the commonly used feeder cells. In particular: HECS increases the recovery of hybridomas after fusion as compared to that in the presence of feeder cells; HECS can substitute feeder cells supporting the growth of hybridomas at the single-cell level under limiting dilution conditions; HECS prolongs the stability of human-mouse hybridomas producing human immunoglobulins (Ig). The active principle which is present in HECS can be different from IL-6. Furthermore the effects of a polyamine derivative, carboxyethyl gamma-aminobutyric acid (CEGABA), were studied on the growth of hybridomas and Epstein-Barr Virus-transformed cell lines. CEGABA enhances the growth of hybridomas through all the steps of hybridoma technology and induces the formation of a large number of EBV-transformed lymphocytes, when added to the culture medium immediately after EBV infection. However, CEGABA shows only a moderate effect on the stability of EBV-transformed cell lines over time, and does not effect the growth of stabilized cell lines. It is possible that CEGABA acts on cells other than EBV-transformed lymphocytes (in fact, after EBV infection all types of mononuclear cells from the blood are present in the culture) and indirectly promotes the growth of EBV-transformed lymphocytes.

A growth factor different from IL-6 promotes the growth of Epstein-Barr virus-transformed cells at low cell density.

Interleukin-6 (IL-6) was recently described to support the growth of Epstein-Barr (EBV) transformed lymphocytes. However no effect was reported on cloning of EBV transformed lymphocytes under limiting dilution conditions. In this paper we demonstrate that the Supernatant of Human Endothelial Cells (HECS) contains a factor(s) different from IL-6 which induces high cloning efficiency of EBV transformed cell lines, cultured under limiting dilution conditions. Furthermore HECS is superior to IL-6 in all the passages of EBV lymphocytes transformation.

Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein. Possible role of cross-reacting antikeratin antibodies.

Antibodies to recombinant heterogeneous nuclear RNP core protein A1 were detected in sera from 27 of 58 patients with rheumatoid arthritis (RA) and from 7 of 31 patients with systemic lupus erythematosus, by immunoblotting and enzyme-linked immunosorbent assay. Protein A1 consists of 2 distinct domains: The N-terminal sequence is identical to a single-stranded DNA binding protein termed UP1, and the C-terminal domain shows a partial homology with keratin. All 7 A1-positive systemic lupus erythematosus sera reacted with UP1, whereas 9 of the 27 A1-positive RA sera did not. In RA, anti-A1 activity was significantly associated with antikeratin antibodies (AKA); these antibodies were present in 23 of 27 A1-positive sera and 10 of 31 A1-negative sera (P less than 0.01). Immunoabsorption with recombinant protein A1 resulted in a significant reduction of AKA titers in 6 of 10 RA sera tested, suggesting that AKA from RA patients may cross-react with the C-terminal portion of the heterogeneous nuclear RNP protein A1.

Autoantibodies to poly(ADP-ribose)polymerase in autoimmune diseases.

Antibodies to various nuclear proteins are frequently found in sera of patients affected by connective tissue diseases and other autoimmune diseases. We investigated the specificity of circulating autoantibodies to poly(ADP-ribose)polymerase (pADPRP) in different autoimmune and connective tissue diseases: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Progressive Systemic Sclerosis (PSS), Sjogren's Syndrome (SS), Undifferentiated Connective Tissue Disease (UCTD), Cryptogenic Fibrosing Alveolitis (CFA) and Sarcoidosis. pADPRP was purified from calf thymus. Antibody specificity was detected by ELISA, western blot and enzyme activity precipitation. Positive values (mean O.D. values + 3 S.D. of 36 normal controls) were obtained in 7/15 SLE patients, 1/18 RA patients, 1/30 PSS, 3/14 SS, 0/5 UCTD, 5/21 CFA and 4/25 Sarcoidosis. The positive sera also recognized the pADPRP protein when tested by western blot. Furthermore the enzyme activity was inhibited after immunoprecipitation by some highly positive sera.

Autoantibodies to DNA topoisomerase II in cryptogenic fibrosing alveolitis and connective tissue disease.

Sera from patients with autoimmune lung and connective tissue diseases were investigated for antibodies to topoisomerase II. Anti-topoisomerase II antibodies were detected by ELISA in 37% of sera from patients with cryptogenic fibrosing alveolitis (CFA), in one (8%) case of sarcoidosis and in 31% of sera from systemic lupus erythematosus (SLE) patients. Sera from rheumatoid arthritis, juvenile rheumatoid arthritis, progressive systemic sclerosis, Sjögren's syndrome and undifferentiated connective tissue disease were negative. CFA and sarcoidosis sera strongly reacted in immunoblotting with a 170 kD protein, also recognized by rabbit antiserum to recombinant topoisomerase II, while SLE sera presented a weak reaction. Pre-adsorption with dsDNA dramatically decreased the topoisomerase II binding in ELISA by the most positive SLE serum, but did not affect the binding by the most positive CFA serum, thus indicating that anti-topoisomerase II reactivity of SLE sera is probably due either to cross-reacting antibodies or, in part, to minor DNA contamination of our enzyme preparation. The determination of DNA topoisomerase II relaxation activity, performed after incubation with antibody-positive sera, showed that only CFA sera precipitate enzymatic activity. The finding that CFA presents antibodies to an enzyme essential to cell survival stresses the role of autoimmunity in the pathogenesis of idiopathic pulmonary fibrosis. This may offer further insight into the cause of autoimmune disease and prove a valuable tool in the study of enzyme molecular biology.

Antibodies to hnRNP core protein A1 in connective tissue diseases.

We investigated the specificity of circulating autoantibodies to a heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), obtained by recombinant DNA technique, in different rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, primary Sjogren's syndrome (SS), idiopathic Raynaud (IR), mixed connective tissue disease (MCTD), and healthy donors. All sera were tested by ELISA on hnRNP A1 protein. Positive values were obtained in 22% SLE, 19% scleroderma, 10% IR, 40% (2/5) MCTD, 5% SS, and 50% RA patients. The majority of patients reacted with the aminoterminal part (UP1) of hnRNP A1; however, some RA patients reacted also with the carboxy-terminal part that shows partial homology with keratin. Therefore, hnRNP A1 (UP1) can be considered a target of antinuclear autoimmunity in various rheumatic disorders.

Anti-SSB/La antibodies in Sjögren's syndrome and related autoimmune diseases. Results of a quantitative immunoassay using a highly purified antigen.

A quantitative immunoassay using a highly purified antigen from HeLa cells has allowed us to detect antibodies to SSB/La in 11/20 patients with primary Sjögren's syndrome (SS), 15/33 with SLE, and 11/35 with progressive systemic sclerosis (PSS). However, positive results were found in only 2/12 patients with idiopathic Raynaud's disease and 2/20 with rheumatoid arthritis, including 4 with secondary SS. Anti-SSB/La were associated with extraglandular manifestations in primary SS, and with a diffuse sclerodermic pattern in PSS. In SS, SLE and PSS, a positive anti-SSB/La test was strongly associated with nodal or spleen enlargement and with an increased level of gamma-globulins. A direct association was also found with positive tests for rheumatoid factors, anti-SSA/Ro and anti-Scl 70. An inverse relationship was however found with anti-nRNP +/- Sm and anticentromere antibodies. Our data suggest that anti-SSB/La antibodies can be regarded as a marker of B-lymphocyte activation in patients with either primary SS, SLE or PSS.

The outer capsid glycoprotein VP7 of simian rotavirus SA11 contains two distinct neutralization epitopes.

Seven neutralizing monoclonal antibodies (MAbs) to the rotavirus simian agent 11 were produced. Although displaying variable degrees of haemagglutination-inhibiting activity, they were shown by radioimmunoprecipitation and Western blot analyses to react with the major outer capsid glycoprotein (VP7). In competition binding assays, MAbs defined two distinct VP7 epitopes, which appeared to be close to each other or partially overlapping. In addition, MAbs of the two epitope groups enhanced binding of a broadly reactive, non-neutralizing, MAb specific for rotavirus group antigen.

Carboxyethyl gamma-aminobutyric acid, a polyamine derivative, improves the recovery of EBV-transformed lymphocytes.

A polyamine derivative, carboxyethyl - Aminobutyric Acid (CEGABA), induces the formation of a large number of Epstein-Barr Virus (EBV) transformed lymphocytes, when added to the culture medium immediately after EBV infection. However, CEGABA shows only a moderate effect on the stability of EBV-transformed cell lines over time, and does not affect the growth of stabilized cell lines. It is possible that CEGABA acts on cells other than EBV transformed lymphocytes (in fact, after EBV infection all types of mononuclear cells from the blood are present in the culture) and indirectly promotes the growth of EBV transformed lymphocytes.