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PubMed1995

Cyclic GMP-dependent protein kinase substrates in rat brain.

Cyclic GMP (cGMP)-dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G-substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion-exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca(2+)-sensitive lipid-binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate-P34-was found. However, after chromatography we detected > 40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP-dependent protein kinase or protein kinase C. Four PKG substrates--P140, P65, P32, and P18--were detected in the cytosol. Six PKG substrates--P130, P85 (doublet), P58, P54, and P38--were enriched from the Ca(2+)-sensitive lipid-binding protein fraction. In peripheral membrane fractions > 30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.

Animals↗

PubMed2003

Inactive hepatic lipase in rat plasma.

Hepatic lipase activity is detectable in liver but also in adrenal glands, ovaries, and plasma. The subunit size of hepatic lipase in liver, adrenal glands, and nonheparin plasma was compared. Hepatic lipase in liver and adrenal glands appeared as a 55 kDa band. In liver, a faint band of lower size was also detected. In nonheparin plasma, hepatic lipase appeared as a doublet of 57 kDa and 59 kDa. When activity/mass ratio was calculated, similar values were obtained for liver and adrenal glands. In plasma this value was much lower. After heparin administration in vivo, hepatic lipase activity in plasma increased nearly 100-fold with appearance of an additional 55 kDa band in postheparin plasma. This band coeluted with activity after preparative polyacrylamide gel electrophoresis. Differences in size persisted after digestion with peptide-N-glycosidase F. A progressive increase in 57 kDa and 59 kDa in postheparin plasma followed disappearance of the 55 kDa band, suggesting that these larger bands originate from the smaller form. In plasma, both smaller and larger forms were associated with HDL, but not with LDL or VLDL. We conclude that rat plasma contains a larger form of hepatic lipase that is inactive in in vitro assay.

Adrenal Glands↗

PubMed1990

Control of endogenous phosphorylation of the major cAMP-dependent protein kinase substrate in adipocytes by insulin and beta-adrenergic stimulation.

In isolated, 32Pi-loaded, rat adipocytes, we have examined phosphorylation of the major cAMP-dependent protein kinase (A-kinase) substrate, a protein that appears to be associated with the lipid storage droplet and migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 65-67-kDa doublet. In control cells, a strong phosphorylation signal is detected as the (+/- cAMP) A-kinase activity ratio ranges from approximately 0.1 to approximately 0.3-0.4 with increasing isoproterenol concentrations. By contrast, insulin-treated cells exhibiting A-kinase activity ratios over the range of 0.1-0.25 contain less 32P in the 65-67-kDa protein than control cells exhibiting identical A-kinase activity ratios. At higher activity ratios (greater than 0.3), this reduction in phosphorylation of the 65-67-kDa protein by insulin disappears. It is concluded that insulin stimulates a phosphatase activity that acts on the 65-67-kDa protein. Insulin actions aside, these studies reveal two interesting phenomena. 1) Whereas elevated, steady-state A-kinase activities are established rapidly (1-2 min) upon isoproterenol stimulation, phosphorylation of the 65-67-kDa substrate proceeds through a burst, followed by a decline to a steady-state level by 10-12 min. An "adaptation" mechanism, providing for a constant response to a constant stimulus, may underlie this lack of parallelism between the time course of phosphorylation and A-kinase activity. 2) Removal of [32Pi] orthophosphate immediately before isoproterenol stimulation leads to a rapid (t approximately 10 min) loss in labeling of the 65-67-kDa protein, whereas the phosphorylation state of other phosphoproteins are not changed. These data suggest that elevation of A-kinase activity leads to a rapid exchange of external Pi with an ATP pool that is used by A-kinase.

Adenosine Triphosphate↗

PubMed2006

Should chemotherapy combinations for advanced non-small cell lung cancer be platinum-based? A meta-analysis of phase III randomized trials.

BACKGROUND: Non-platinum regimens have been proposed as an alternative to the platinum-based combinations for treatment of advanced non-small cell lung cancer. However, conflicting results were reported. METHODS: Meta-analysis of phase III trials randomizing platinum-based versus non-platinum combinations as first-line chemotherapy with 1-year survival rate as a primary endpoint. Fourteen trials have been identified. Experimental arms were gemcitabine/vinorelbine (n=4), gemcitabine/taxane (n=7), gemcitabine/epirubicin (n=1), paclitaxel/vinorelbine (n=1), and gemcitabine/ifosfamide (n=1). The comparator was a doublet of a platinum compound plus a third generation agent for all but two studies (triplets). Updated data were available for 13 studies. The Peto and Yusuf method was used to generate odds ratios (OR). All tests are two-sided. RESULTS: A statistical heterogeneity was detected when the 13 studies were analyzed. Considering that current guidelines recommend platinum-based doublets as standard therapy we therefore limited the meta-analysis to the set of 11 phase III studies which used a platinum-based doublet (2298 and 2304 patients in platinum-based and non-platinum arms, respectively). No significant heterogeneity was detected in this consistent group of studies. Patients treated with a platinum-based regimen benefited from a statically significant reduction in the risk of death at 1 year (OR: 0.88, 95% CI 0.78-0.99; p=0.044) and a lower risk of being refractory to chemotherapy (OR: 0.87, 0.73-0.99; p=0.049). Forty-four (1.9%) and 29 (1.3%) toxic-related deaths were reported for platinum-based and non-platinum regimens, respectively (OR: 1.53; 0.96-2.49, p=0.08). An increased risk of grade 3-4 gastro-intestinal and hematological toxicity for patients receiving platinum-based chemotherapy was statistically demonstrated. There was no statically significant increase in risk of febrile neutropenia, OR=1.23 (0.94-1.60, p=0.063). CONCLUSION: A platinum-based doublet induced a statically significant reduction in the risk of death when compared with non-platinum chemotherapy without inducing an unacceptable increase in toxicity.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors.

In adrenocortical tumors, malignancy is strongly associated with insulin-like growth factor II (IGF-II) gene overexpression and abnormalities at the 11p15 locus, suggesting a role for this growth factor in adrenocortical tumorigenesis. To further investigate this role, the IGF/IGF-binding protein (IGFBP) system was analyzed in 18 adrenocortical tumors, classified into 2 groups on the basis of their IGF-II messenger ribonucleic acid (mRNA) content (group 1, normal IGF-II mRNA content, mostly benign tumors; group 2, high IGF-II mRNA content, mostly malignant tumors). Group 2 tumors contained 10 times more IGF-II protein than group 1 tumors or normal adrenal tissue (P < 0.001), indicating efficient translation of IGF-II mRNA in malignant tumors. Western ligand blotting detected various functional IGFBPs in normal adrenocortical glands and tumors: a doublet of 39-42 kDa identified by immunoblotting as IGFBP-3, a band at 32 kDa, and bands at 29-30 and 24 kDa. Total IGFBP-3 protein levels were similar in the two groups of tumors. By contrast, malignant tumors differed from benign ones by specific expression of the 32-kDa IGFBP. Immunoblotting identified this 32-kDa band together with a proteolytic fragment of 25 kDa as IGFBP-2, and quantitative analysis showed significantly higher levels of total IGFBP-2 in malignant tumors than in benign tumors (P < 0.001). Despite enhanced levels of IGBP-2 protein in malignant tumors, no increase in IGFBP-2 mRNA levels was detected, suggesting post-transcriptional regulation of this IGFBP. These results confirm the major role of IGF-II in adrenocortical tumorigenesis and suggest that IGFBP-2 may be a regulator of IGF-II proliferative effects in this tumor system.

Adolescent↗

PubMed1981

Impaired erythrocyte doublet formation in diabetes.

A new means by which to examine erythrocyte deformability, the rate at which erythrocytes in dilute suspension form doublets after settling to the surface of a microscope slide, has been developed and tested. Doublet formation consists of the evaluation and subsequent apposition of one cell over another, a process limited by the ability of each red cell membrane to bend. The cell-cell attraction that promotes doublet formation is controlled by adding an appropriate amount of dextran to the artificial suspending medium. Videotaping permits careful analysis of doublet formation rate and maintains a permanent record. When erythrocytes from 20 diabetic and 20 non-diabetic subjects were studied, doublet formation rates were found to be strikingly different. Cells from half of the diabetics studied formed less than three doublets in 20 min while the non-diabetic mean for the same period was 15 doublets. No overall correlation between doublet formation and fasting glucose could be found. No relation between doublet formation rate and type of diabetes, treatment, or microvascular complications was observed. Doublet formation rate is a simple and rapid means of detecting and studying reduced erythrocyte deformability in diabetes.

Adult↗

PubMed1991

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.

Antibodies, Monoclonal↗

PubMed2005

Decreased glutathione transferase levels in rd1/rd1 mouse retina: replenishment protects photoreceptors in retinal explants.

Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.

Animals↗

PubMed1997

Histone H1 expression varies during the Leishmania major life cycle.

The deduced amino acid sequence of Leishmania major sw3 cDNA reveals the presence of characteristic histone H1 amino acid motifs. However, the open reading frame is of an unusually small size for histone H1 (105 amino acids) because it lacks the coding potential for the central hydrophobic globular domain of linker histones present in other eukaryotes. Here, we provide biochemical evidence that the SW3 protein is indeed a L. major nuclear histone H1, and that it is differentially expressed during the life cycle of the parasite. Due to its high lysine content, the SW3 protein can be purified to a high degree from L. major nuclear lysates with 5% perchloric acid, a histone H1 preparative method. Using an anti-SW3 antibody, this protein is detected as a 17 kDa or as a 17/19 kDa doublet in the nuclear subfraction in different L. major strains. The nuclear localization of the SW3 protein is further supported by immunofluorescence studies. During in vitro promastigote growth, both the sw3 cytoplasmic mRNA and its protein progressively accumulate within parasites from early log phase to stationary phase. Within amastigotes, the high level of H1 expression is maintained but decreases when amastigotes differentiate into promastigotes. Together, these observations suggest that the different levels of this histone H1 protein could influence the varying degrees of chromatin condensation during the life-cycle of the parasite, and provide us with tools to study this mechanism.

Amino Acid Sequence↗

PubMed1987

Identification of clinical and environmental isolates of Legionella pneumophila by analysis of outer-membrane proteins, ubiquinones and fatty acids.

47 strains of L. pneumophila, 9 reference strains of eight serogroups, 15 clinical isolates and 23 water-derived strains from different cities were analysed with respect to three chemical constituents of the cell envelope: outer- membrane proteins (OMPs), ubiquinones and fatty acids. The OMPs were obtained by Sarkosyl-extraction of a total membrane preparation and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ubiquinone content was quantitated by high-performance liquid chromatography (HPLC) and the fatty acid composition was established by gas-liquid chromatography coupled with mass spectrometry (GLC-MS). In all strains the OMP yielded the known characteristic pattern with the major component of 29 kilodalton. The ubiquinone content was very consistent within different strains revealing Q-12 with 65% as the predominant feature. Q-11 and Q-13 were present in amounts of 11% and 23%, respectively. Q-14 was found in trace amounts of about 0.65%. Only a very few ubiquinones with side chains shorter than 11 residues were present. GLC-MS allowed an unequivocal identification of each fatty acid. The branched chain fatty acid i-16:0 was the predominant feature with 33% followed by the unsaturated straight-chain acid 16:1. In this respect earlier reports by other authors were confirmed but minor compounds not detected so far could be revealed as 15:1, the doublet 18:1 and i-18:0, 19:1 and 20:1. OMP profiles are easy to establish and to interpret. They are well suited for identification of the species L. pneumophila. Ubiquinone analysis permits rapid but preliminary species identification, whereas fatty acid analysis by GLC-MS is an intricate technique providing a valuable chemical marker for L. pneumophila. The biochemical analyses presented here can supplement immunological studies, clarify immunological findings and help to identify new serogroups of L. pneumophila.

Bacterial Outer Membrane Proteins↗

PubMed2003

Squid visual arrestin: cDNA cloning and calcium-dependent phosphorylation by rhodopsin kinase (SQRK).

Arrestin binding to rhodopsin is one of the major mechanisms of termination of photoresponses in both vertebrates and invertebrates. Here we report the cDNA cloning and characterization of a 48-kDa visual arrestin from squid (Loligo pealei). The cDNA encoded a protein that had 56-64% amino acid sequence similarity to reported arrestin sequences. This protein does not encode any distinct modular domains but contains five fingerprint regions that have been identified within arrestins. Antibodies raised to the recombinant arrestin protein detected arrestin expression only in the eye and recognized a doublet in photoreceptor membranes, representing unphosphorylated and phosphorylated arrestin. In squid eye membranes, arrestin was phosphorylated in a Ca2+-dependent manner and this phosphorylation was inhibited by antibodies raised against squid rhodopsin kinase, but not by inhibitors of protein kinase C or calmodulin kinase. Addition of purified squid rhodopsin kinase to washed rhabdomeric membranes resulted in phosphorylation of rhodopsin, and arrestin was also phosphorylated when calcium was present. This is the first report of a rhodopsin kinase phosphorylating an arrestin substrate, and suggests a dual role for this kinase in the inactivation of the squid visual system.

Amino Acid Sequence↗

PubMed1990

Persistent induction of cyclooxygenase in p60v-src-transformed 3T3 fibroblasts.

A BALB/c 3T3 cell line infected with the temperature-sensitive Rous sarcoma virus strain LA90 has been used to investigate early, p60v-src-dependent changes in gene expression (protein synthesis). Giant two-dimensional electrophoresis, which can resolve greater than 3000 polypeptides from [35S]methionine-labeled cell lysates, was used to detect the induction of a p72-74 (72-74 kDa) doublet (pI 7.5) after activation of p60v-src at 35 degrees C. Antiserum against cyclooxygenase (prostaglandin synthase or prostaglandin endoperoxide synthase) specifically immunoprecipitated the p72-74 doublet. The p72-74 doublet was also induced by platelet-derived growth factor and by phorbol 12-myristate 13-acetate and was elevated in an NIH 3T3 cell line transformed by wild-type src. Activation of p60v-src caused a persistent increase in p72-74, whereas the effect of the growth factor was transient. These dissimilar kinetics of induction were paralleled by changes in cyclooxygenase activity. Down-regulation of protein kinase C inhibited subsequent induction of cyclooxygenase by phorbol myristate acetate but did not block induction by p60v-src. The glucocorticoid agonist dexamethasone inhibited induction of cyclooxygenase by p60v-src. Although induction of this enzyme may not be directly involved in transformation, the data support the view that oncogenic transformation may result, not from expression of transformation-specific genes, but from persistent changes in the expression of genes normally induced only transiently during passage from the G0 stage of the cell cycle.

Animals↗

PubMed1988

Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli.

The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated. Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units. Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors. Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity. Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria. Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100%. Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum. The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting. The predominant component was always the 32-kDa polypeptide. We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E. coli.

Acetylgalactosamine↗

PubMed1987

Visna virus exhibits a complex transcriptional pattern: one aspect of gene expression shared with the acquired immunodeficiency syndrome retrovirus.

A complex pattern of gene expression was found for visna virus in a highly permissive cell culture system in vitro. In addition to the genomic RNA (9.4 kilobases [kb]), five other mRNAs were detected. The three large RNA transcripts (5.0 kb and a doublet at 4.3 kb) arise by a single splicing event joining 5' sequences to sequences located at positions 3' to the pol gene. The two smallest transcripts (1.8 and 1.5 kb) are at least doubly spliced mRNAs which contain sequences derived from the 5' end of the genome, the region between the pol and env genes, and 3' terminal sequences. In addition to this complex pattern of transcription, the mRNAs appear to be regulated temporally. The 1.5-kb mRNA appears 6 h later than the other transcripts. The significance of this complex pattern of gene expression in the unique aspects of the lentivirus life cycle and pathogenesis is considered.

Endonucleases↗

PubMed2004

Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin.

Antibodies directed to the Sa antigen are highly specific for rheumatoid arthritis (RA) and can be detected in approximately 40% of RA sera. The antigen, a doublet of protein bands of about 50 kDa, is present in placenta and in RA synovial tissue. Although it has been stated that the Sa antigen is citrullinated vimentin, experimental proof for this claim has never been published. In this study, we investigated the precise nature of the antigen. Peptide sequences that were obtained from highly purified Sa antigen were unique to vimentin. Recombinant vimentin, however, was not recognized by anti-Sa reference sera. In vivo, vimentin is subjected to various post-translational modifications, including citrullination. Since antibodies to citrullinated proteins are known to be highly specific for RA, we investigated whether Sa is citrullinated and found that Sa indeed is citrullinated vimentin. Anti-Sa antibodies thus belong to the family of anticitrullinated protein/peptide antibodies. The presence of the Sa antigen in RA synovial tissue, and the recent observation that vimentin is citrullinated in dying human macrophages, make citrullinated vimentin an interesting candidate autoantigen in RA and may provide new insights into the potential role of citrullinated synovial antigens and the antibodies directed to them in the pathophysiology of RA.

Antibodies, Anti-Idiotypic↗

PubMed1999

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis.

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.

Adolescent↗

PubMed1993

Characterization of interleukin-7-induced changes in tyrosine phosphorylation and c-myc gene expression in normal human T cells.

Interleukin-7 (IL-7) is a growth factor involved in regulating lymphopoiesis. We have chosen to study the signal transduction pathway of IL-7 in normal human peripheral blood T lymphocytes. Two early events that occur as a consequence of specific ligand-receptor interaction were examined: activation of protein tyrosine kinases and induction of primary response gene expression. Following treatment of human peripheral blood T cells with IL-7, four cellular proteins with relative molecular weights of 95- (doublet), 105-, and 130-kd were rapidly tyrosine phosphorylated as detected by immunoblotting with an antiphosphotyrosine monoclonal antibody (MAB). The 105-kd tyrosine-phosphorylated protein was membrane-associated after IL-7 stimulation. Treatment of human peripheral blood T cells with IL-7 enhanced expression of the primary response gene c-myc approximately three-fold, as detected by Northern blotting, in the presence or absence of protein synthesis. The rate of c-myc gene transcription increased in the presence of IL-7 and could account for the observed elevation of c-myc RNA levels. In addition, IL-7 treatment induced a slight increase in c-myc message stability. Experiments performed with the protein tyrosine kinase inhibitor genistein and the serine-threonine kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) demonstrated that these kinases were required for IL-7 enhancement of c-myc expression. Treatment with tetradecanoyl phorbol acetate (TPA), a potent activator of protein kinase C (PKC), in combination with IL-7 induced a level of c-myc expression greater than that elicited by either factor alone, suggesting that TPA and IL-7 utilize cooperative signaling pathways to increase c-myc gene expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Autoantibody to DNA topoisomerase II in primary liver cancer

A patient with chronic hepatitis associated with hepatitis C virus infection was observed to convert from antinuclear antibody-negative to antinuclear antibody-positive status at the time when liver cancer was detected. The newly recognized antibodies reacted with a nuclear protein doublet of 170 and 180 kDa in immunoblotting, and in fluorescence-activated flow cytometry the antigens were shown to vary in expression level in a cell cycle-related manner: minimum in G1, increasing in S, and maximum in G2 and M. In synchronized HeLa and HEp-2 cells, immunofluorescence microscopy showed uniformly distributed staining of the nucleoplasm in S-phase, with increased intensity of nucleoplasmic staining in G2, at which time nucleolar staining was also present. In M, condensed chromosomes were uniformly stained. Using previously characterized polyclonal antibodies to DNA topoisomerase II (topo II) as reference markers, the antigens recognized by the patient's serum were shown in Western blotting to have the same mobilities as DNA topo IIalpha (170 kDa) and beta (180 kDa) isoforms. The patient's serum was also highly efficient in inhibiting DNA topo II in an in vitro functional assay. Antibody to DNA topo II appeared de novo in close association with transformation to cancer, and since dysregulation of DNA topo II is considered to be involved in some forms of tumorigenesis, the observed antibody response in this patient could conceivably be an immune reaction to the abnormally regulated protein.

Journal Article↗