p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis.

DNA degradation is one of the biochemical hallmarks detected in apoptotic cells, and several nucleases have been reported to function cooperatively in this process. It has also been suggested that different sets of nucleases are activated by different stimuli, and induce distinct patterns of DNA degradation. Here we report that apoptosis-enhancing nuclease (AEN) is a novel direct target gene of p53. AEN is induced by p53 with various DNA damage, and its expression is regulated by the phosphorylation status of p53. We demonstrate that AEN is a typical exonuclease with conserved exonuclease domains Exo I-III, and it targets both single- and double-stranded DNA and RNA. AEN induces apoptosis by itself, and the conserved domains are essential for both AEN nuclease activity and its apoptosis-inducing ability. AEN possesses nuclear and nucleolar localization signals, and it translocates from the nucleolus to nucleoplasm upon apoptosis induction. We also show the dislocation of nucleophosmin in conjunction with the translocation of AEN to the nucleoplasm, indicating the ability of AEN in nucleolus disruption. In addition, AEN is shown to be required for efficient DNA fragmentation in p53-dependent apoptosis. These results suggest that AEN is an important downstream mediator of p53 in apoptosis induction.

Angiotensin-converting enzyme inhibition improves cardiac fatty acid metabolism in patients with congestive heart failure.

This study aimed to examine whether angiotensin-converting enzyme (ACE) inhibition improved cardiac fatty acid metabolism in patients with congestive heart failure (CHF). Myocardial 123I-beta-methyl-iodophenylpentadecanoic acid (123I-BMIPP) imaging was performed in 25 patients with CHF and in 10 control subjects. Myocardial 123I-BMIPP images were obtained 30 min and 4 h after tracer injection. The heart-to-mediastinum (H/M) ratio of 123I-BMIPP uptake and the washout rate of 123I-BMIPP from the myocardium were calculated. Patients were given enalapril for 6 months, and 123I-BMIPP imaging was repeated. H/M ratios on early and delayed images were lower in CHF patients than in normal controls (P<0.01). The washout rate of 123I-BMIPP from the myocardium was faster in CHF patients than in controls (P<0.01). As the severity of the New York Heart Association (NYHA) functional class increased, the H/M ratio decreased and the washout rate increased. The washout rate of 123I-BMIPP was inversely correlated with left ventricular fractional shortening (R=-0.62, P<0.01). ACE inhibition with enalapril increased the H/M ratio on delayed images (P<0.05) and reduced the washout rate of 123I-BMIPP (P<0.05) in CHF patients. These data suggest that: (1) angiotensin II-mediated intracellular signalling activation may be a possible mechanism for the decreased myocardial uptake and enhanced washout of 123I-BMIPP in heart failure patients; and (2) the improvement in fatty acid metabolism by ACE inhibition may represent a new mechanism for the beneficial effect of this therapy in heart failure.

Pulsed Doppler tissue imaging for the assessment of myocardial viability: comparison with 99mTc sestamibi perfusion imaging.

The aim of the present study was to examine whether Doppler tissue imaging demonstrated comparable diagnostic performance for the detection of viable myocardium compared to myocardial perfusion imaging with Tc hexakis-2-methoxyisobutylisonitrile (MIBI). We studied 30 patients with old myocardial infarction who underwent percutaneous transluminal coronary angioplasty (PTCA). Myocardial single photon emission computed tomography (SPECT) with Tc-MIBI and two-dimensional echocardiography were carried out within 7 days before PTCA. We measured regional Tc-MIBI uptake for each myocardial segment from SPECT and peak systolic velocity and a ratio of regional pre-ejection period to regional ejection time (PEP/ET) from pulsed Doppler tissue imaging. Biplane left ventriculography was performed before interventional procedures and repeated 3 months after PTCA. Myocardial viability was determined when wall motion was improved at least one grade after PTCA. The peak systolic velocity was positively correlated with regional Tc-MIBI uptake (R =0.59, P<0.01). The PEP/ET demonstrated inverse correlation with Tc-MIBI uptake ( R=-0.59, P<0.01). Peak systolic velocity of viable segments was higher than that of non-viable segments ( P<0.05). The PEP/ET was lower in viable segments than in non-viable segments ( P<0.05). Peak systolic velocity and PEP/ET demonstrated high diagnostic accuracy for detecting viable myocardium compared with Tc-MIBI perfusion imaging (80% and 79% vs 90%). These data indicate that measurements of regional peak systolic velocity and PEP/ET by Doppler tissue imaging are useful for evaluating myocardial viability quantitatively and provide helpful information for a clinical judgment in an interventional strategy.

Smoking behavior, initiating and cessation factors among Japanese nurses: a cohort study.

The prevalence of smoking among Japanese nurses, specially in their twenties, is higher than that among the general female population. To examine smoking behavior, smoking initiating and cessation factors, we conducted a cohort study through questionnaire survey, targeting nurses (n=1572) working at 11 hospitals located in Tokyo metropolitan area. The first survey was conducted using a confidential questionnaire on smoking, followed by a second survey conducted in the same manner on the same subjects two years later. As to smoking status after two years, 8% (95%CI=1.5%) started smoking and 6% (95%CI=1.4%) quitted resulting in a 2% increase in the prevalence of current smoking. The average nicotine dependence for nurses who were smokers in the two surveys rose from 3.9 to 4.3 (P<0.05). Smoking behavior of mother, friends, or superiors at work had a significant influence on smoking behavior of nurses. As to smoking cessation factors, the idea that women and medical workers should not smoke, and living with family each had a significant influence. Considering the fact that 6% of nurses in this study succeeded in quitting smoking within two years, it is required that anti-smoking education be conducted at medical institutions to decrease the prevalence of current smoking among the nurses in Japan.

Mitotic specific phosphorylation of serine-1212 in human DNA topoisomerase IIalpha.

It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.

[The validity of repeated mail surveys concerning smoking habits for Japanese physicians].

PURPOSE: A cross-sectional study of smoking prevalence among medical doctors was performed using questionnaires. Mailing four times proved essential in order to obtain a reasonable number of responses from the subjects. The objective of the present study was to analyze the smoking characteristics of the subjects who returned the questionnaires after the second to fourth mailings. METHODS: A set of an anonymous questionnaire and a letter from the President of the Japan Medical Association (JMA) requesting cooperation was mailed with a return envelope with the subject's name and address written to 3,000 male and 1,500 female doctors randomly selected from the roster membership of the JMA. The survey was conducted between February and June 2000. RESULTS: The collection rate after the first mailing was 66%, while the subtotal collection rate for the second through fourth mailings was 21%, bringing the grand-total to 87%. The total prevalence of smoking among the subjects who had sent back the questionnaire on the second to fourth mailings was approximately 1.5 times higher than for those who had sent back the questionnaire after the initial mailing for both male and female subjects. As to other characteristics of the subjects who only responded after the second to fourth mailings were: working in hospitals (odds ratios; male: 1.39, female: 1.47), not giving smoking cessation guidance (odds ratio; male: 0.58), and not recognizing the idea that doctors should not smoke (odds ratio; female: 0.67). CONCLUSIONS: The results suggested that for future surveys on smoking to be conducted in academic institutions or work-places, efforts to obtain responses from those who do not answer the first attempt should take into consideration these points.

[The influence of lifestyle and night-shift work on sleep problems among female hospital nurses in Japan].

STUDY OBJECTIVE: In this study, by conducting a questionnaire survey, we aimed to clarify the situation regarding sleep disorders in female hospital nurses and their relation with night-shift work and lifestyle. METHODS: The subjects were female nurses working at 5 hospitals, each with more than 400 beds. The survey was carried out in July 2000. The questionnaire contained six items concerning sleep quality from the Pittsburgh Sleep Quality Index (PSQI), two new items on sleep drafted by ourselves, and some questions on lifestyle and shift-work status. RESULTS: Among all female nurses, statistically significant differences were observed between those working and those not working night shifts for 7 items regarding sleep (P < 0.05). Significant correlations were observed between sleep disorders and the following factors: (1) working night shift, (2) having anxiety or stress, (3) getting less than 6 hours of sleep, (4) working in cities, (5) having children, and (6) bathing more than 1 hour before going to bed. In addition, significant correlations were observed between getting less than 6 hours of sleep and the following factors: (1) being 40 years of age or older, (2) working in cities, and (3) having anxiety or stress. CONCLUSIONS: The results of this study suggest that sleep problems among nurses are associated not only with night-shift work but also with lifestyle. They also suggest that nurses who work night shifts, especially in Tokyo, should try to get sufficient hours of sleep to ensure good quality of sleep.

Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis.

Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.

In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4.

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.

Standard radial bone mineral density and physical factors in ordinary Japanese women.

To prevent osteoporosis, which is expected to increase in incidence in this rapidly aging society, in recent years bone mineral density (BMD) has frequently been measured as a predisposition index. However, these measurements are made on different sites with different apparatus, and the results are independently studied by different institutions. In our present investigation, to establish the standard radius BMD as determined by dual-energy X-ray absorptiometry (DXA), we carried out a general population survey in 29 municipalities and prefectures on 11,252 locally residing females aged 15 to 83 years (mean, 35.61 +/- 12.85 years). Their YAM (young adult mean) BMD was estimated at 0.664 +/- 0.054 g/cm2, which was almost the same as the figure given in the 1996 version of the diagnostic criteria for primary osteoporosis. We further studied the relationships of BMD to age and physical factors known to be influential to BMD. It was found that BMD was correlated negatively to age and positively to body mass index (BMI). The average values we obtained for age and physique groups appeared to have provided reliable indices for the primary prevention of osteoporosis.

Enhanced expression and shedding of tumor necrosis factor (TNF) receptors from mononuclear leukocytes in human heart failure.

TNFalpha exerts its functions by engaging two receptors (TNF-RI and TNF-RII). The extracellular parts of the receptors are proteolytically shed to the soluble forms by a matrix metalloproteinase-like enzyme (sTNF-RI and sTNF-RII). The soluble TNF receptors can neutralize TNFalpha activities. Circulating levels of both sTNF-RI and sTNF-RII are elevated in patients with congestive heart failure (CHF). It remains unclear how a large amount of sTNF-RI and sTNF-RII is mobilized into the circulation. Mononuclear leukocytes were obtained from 14 controls and 21 patients with CHF. TNF-RII of the cells from CHF patients was upregulated in the cell-surface expression and mRNA transcripts. Besides enhanced shedding of TNF-RII on the cells from CHF patients with phorbol myristate acetate (PMA), sera from CHF induced shedding of TNF-RII on the cells from normal volunteers. Thus, the enhancement of both expression and shedding of TNF-RII may be related to increased circulating levels of the soluble TNF receptor in patients with CHF. The presence of CHF may affect the regulation of TNF receptors, which may modulate the responsiveness to TNFalpha in the tissues of patients with CHF.

Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity.

IL-2-activated NK cells exhibit cytotoxic activity against a wide variety of tumor cells in a non-MHC-restricted fashion and in the absence of prior sensitization. The molecular mechanisms that regulate the cytotoxicity and attachment of activated killer cells to tumor target cells are not known. We provide genetic evidence in CD44(-/-) and LFA-1(-/-) mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cells. This defect in cytotoxicity was significantly enhanced in mice that carried a double mutation of both CD44 and LFA-1. In vitro differentiation, TNF-alpha and IFN-gamma production, and expression of the cytolytic effector molecules perforin and Fas-L were comparable among IL-2-activated NK cells from LFA-1(-/-), CD44(-/-), CD44(-/-)LFA-1(-/-), and control mice. However, CD44(-/-), LFA-1(-/-), and CD44(-/-)LFA-1(-/-) IL-2-activated NK cells showed impaired binding and conjugate formation with target cells. We also show that hyaluronic acid is the principal ligand on tumor cells for CD44-mediated cytotoxicity of IL-2-activated NK cells. These results provide the first genetic evidence of the role of adhesion receptors in IL-2-activated NK killing. These data also indicate that distinct adhesion receptors cooperate to mediate binding between effector and target cells required for the initiation of "natural" cytotoxicity.

Soluble tumor necrosis factor receptors are elevated in relation to severity of congestive heart failure.

The level of tumor necrosis factor alpha (TNF-alpha) is increased in patients with congestive heart failure and may play an important role in the development and progression of heart failure. Two types of TNF receptor (TNF-RI and TNF-RII) are expressed in virtually every cell and have different biologic roles. Soluble forms of the two receptors (sTNF-RI and sTNF-RII) have been identified as extracellular domain fragments. Serum levels of TNF-alpha, sTNF-RI and sTNF-RII were measured in 66 patients with heart failure and 27 control subjects using an enzyme-linked immunosorbent assay (ELISA). Hemodynamic variables, norepinephrine, atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) were evaluated. TNF-alpha was significantly higher in patients with heart failure than in controls subjects (9.4 +/- 1.4 vs 4.8 +/- 0.8 pg/ml; p < 0.05). sTNF-RI and -RII were significantly increased in relation to the severity of heart failure (control subjects, 0.66 +/- 0.04 and 1.97 +/- 0.15 ng/ml; NYHA class II, 1.10 +/- 0.08 and 2.28 +/- 0.12 ng/ml; NYHA class III, 1.63 +/- 0.22 and 3.00 +/- 0.24 ng/ml; NYHA class IV, 2.78 +/- 0.46 and 4.52 +/- 0.62 ng/ml, respectively). In 9 patients whose clinical symptoms improved after treatment, the levels of sTNF-RI and -RII decreased by 17.3 +/- 5.7% (p < 0.05) and 22.1 +/- 6.9% (p < 0.05), respectively. There were significant positive correlations between sTNF-RI and -RII and mean pulmonary pressure (r = 0.69 and r = 0.61; p < 0.001) and mean capillary wedge pressure (r = 0.65 and r = 0.54; p < 0.001 and p < 0.01, respectively), but not with left ventricular end-diastolic volume or ejection fraction (NS). sTNF-RI and -RII were also significantly positively correlated with plasma levels of norepinephrine (r = 0.75 and r = 0.50; p < 0.001 and p < 0.05), ANP (r = 0.72 and r = 0.70; p < 0.001), and BNP (r = 0.60 and r = 0.60; p < 0.001). In conclusion, soluble TNF receptors are increased in proportion to the severity of congestive heart failure and may reflect the current status of congestive heart failure rather than the level of left ventricular dysfunction.

Serum levels of soluble form of Fas molecule in patients with congestive heart failure.

Compared with soluble Fas molecule (sFas, an inhibitor of Fas-mediated apoptosis) in normal volunteers, the serum level of sFas significantly increased by 41% in New York Heart Association (NYHA) class III (p <0.05) and by 97% in NYHA class IV patients with congestive heart failure (p <0.001). Furthermore, sFas showed correlations with soluble forms of TNF receptor-p55 (RI) and -p75 (RII) (r = 0.68 and r = 0.56) which inhibit activities of TNF alpha.

Roles of nitric oxide and adenosine in the regulation of coronary conductance in the basal state and during reactive hyperemia.

Nitric oxide (NO) and adenosine are important mediators in the regulation of coronary vascular tone and are released into the interstitium from the vascular endothelium and myocardium, respectively. The roles of these autacoids in the regulation of coronary flow in the basal and reactive hyperemic states were examined in Langendorff rabbit hearts perfused with oxygenated Krebs-Henseleit solution at 37 degrees C and 110 mmHg pressure. Instantaneous perfusion pressure-flow relationships were analyzed to derive coronary conductance both in the basal state and during the early phase of reperfusion (hyperemic state). N omega-nitro-L-arginine methyl ester (L-NAME) at increasing concentrations (10(-6) to 10(-4) mol/L) (n = 7) and 8-phenyltheophylline (8-PT) at increasing concentrations (10(-9) to 10(-6) mol/L) (n = 7) were applied to assess the role of NO and adenosine, respectively. L-NAME dose-dependently reduced the coronary conductance in both the basal and early hyperemic states, while 8-PT dose-dependently reduced conductance only in the hyperemic state. Changes in conductance during the early hyperemic phase correlated well with changes in the debt repayment ratio for either L-NAME (r = 0.94) or 8-PT (r = 0.99). These data suggest that a flow-related NO release mechanism regulates the coronary conductance in both the basal and hyperemic states while the metabolic regulation of adenosine release plays a role in the presence of ischemia.

Immunoaffinity purification and characterization of CACGTG sequence-binding proteins from cultured mammalian cells using an anti c-Myc monoclonal antibody recognizing the DNA-binding domain.

c-Myc protein contains a basic helix-loop-helix and leucine zipper (bHLHLZ) structure in its carboxyl terminal region, forms heterodimers with Max, and binds to the CACGTG sequence in DNA. A number of bHLHLZ proteins are present in cells, and some of them bind to the same DNA sequence. Using an anti c-Myc monoclonal antibody, MYC5, raised against a bacterially synthesized c-Myc protein, we have carried out immunoaffinity purification of c-Myc proteins from cultured mammalian cells. The immunoaffinity-purified fraction was found to contain not only c-Myc but also other CACGTG sequence-binding proteins, such as Max, Mad, and USF, indicating a wide cross-reaction to CACGTG sequence-binding proteins. The MYC5 antibody recognized the common structural motif in their basic region required for sequence-specific DNA binding and was shown to inhibit their DNA-binding activities. The immunoaffinity-purified N-Myc, Max, Mad, and, presumably, c-Myc were highly phosphorylated, and phosphatase treatment increased the DNA-binding activity of Myc, suggesting that the DNA-binding activity of c-Myc was regulated by phosphorylation in vivo. From these results, we can conclude that the MYC5 antibody constitutes a powerful tool for the purification and characterization of c-Myc and other CACGTG sequence-binding proteins.

The distribution and expression of the two isoforms of DNA topoisomerase II in normal and neoplastic human tissues.

In mammalian cells, there are two isoforms of DNA topoisomerase II, designated alpha (170-kDa form) and beta (180-kDa form). Previous studies using cell lines have shown that the topoisomerase IIalpha and beta isoforms are differentially regulated during the cell cycle and in response to changes in growth state. Moreover, both isoforms can act as targets for a range of anti-tumour drugs. Here, we have analysed the normal tissue distribution in humans of topoisomerase IIalpha and beta using isoform-specific antibodies. In addition, we have studied expression of these isoforms in 69 primary tumour biopsies, representative either of tumours that are responsive to topoisomerase II-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase IIalpha was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (e.g. high-grade lymphomas and seminomas), there was a high level of topoisomerase IIalpha, although expression was still detectable in colon tumours, indicating that expression of this isoform is not sufficient to explain the intrinsic drug resistance of colon tumours. Topoisomerase IIbeta was expressed ubiquitously in vivo and was localized in both the nucleoli and the nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues. We conclude that topoisomerase IIalpha is a strict proliferation marker in normal and neoplastic cells in vivo, but that topoisomerase IIbeta has a much more general cell and tissue distribution than has topoisomerase IIalpha. The apparent up-regulation of topoisomerase IIbeta in neoplastic cells has implications for the response of patients to anti-tumour therapies that include topoisomerase II-targeting drugs.

Threonine 1342 in human topoisomerase IIalpha is phosphorylated throughout the cell cycle.

To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase IIalpha containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase IIalpha immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase IIalpha mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase IIalpha through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase IIalpha and DNA and analyzed by flow cytometry. Cells in the G2-M phase contained about double the PT1341-reacted topoisomerase IIalpha than did cells in G1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase IIalpha antibody. Thus, threonine 1342 in topoisomerase IIalpha is phosphorylated throughout the cell cycle.

Degradation of topoisomerase IIalpha during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system.

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.

Immunohistochemical study of DNA topoisomerase II in human gastric disorders.

Topoisomerase II (topo II) separates chromosomes at the end of mitosis and is also the target for various chemotherapeutic agents. Expression of this enzyme has been demonstrated to increase rapidly at the end of the S to G2/M phase and decrease after the completion of mitosis. We immunolocalized topo II in specimens of both normal and neoplastic human gastric mucosas to evaluate expression of this enzyme. Three different antibodies were used for the immunostaining of topo II (anti-topo II alpha isoform, anti-topo II beta isoform and anti-topo II alpha and -beta isoforms). There were no significant differences in topo II labeling index (LI) between frozen and paraffin-embedded tissue sections obtained from the same cases. Topo II LI was significantly correlated with Ki67 LI in all of the specimens examined. The area of cells positive for Topo II was much narrower than that of Ki67 in the normal gastric glands, and the pattern of Topo II immunolocalization in both adenomas and adenocarcinomas was also essentially the same as that of Ki67. The topo II LI values (positive cells/1000 cells) for normal gastric gland, adenoma, intestinal-type adenocarcinoma, and diffuse-type adenocarcinoma were 114.7 +/- 2.2, 266.7 +/- 18.8, 277.6 +/- 19.2, and 324.5 +/- 5.3, respectively. Significant differences in topo II LI and topo II/Ki67 index were observed between normal and neoplastic mucosas (P < 0.0001) and between adenomas or intestinal-type adenocarcinoma and diffuse-type adenocarcinoma (P < 0.001 and P < 0.01, respectively). Simultaneous measurement of topo II alpha and nuclear DNA content by two-parameter flow cytometry revealed that the Jurkat cell line established from acute lymphocytic leukemia cells expressed the enzyme in cells at other than S and G2/M phases of the cell cycle whereas topo-II alpha-positive cells were predominantly observed in S and G2/M phases of the cell cycle in the cells from normal lymph nodes. These findings suggest that dys-regulation or qualitative changes of topo II alpha expression are associated with malignancy. Topo II immunostaining can thus detect proliferating cells in routinely processed tissue sections and can indicate the altered topo II alpha expression in human cancers, which may be related to the sensitivity to topo-II-targeted chemotherapeutic agents.