Induction of topoisomerase II activity after ErbB2 activation is associated with a differential response to breast cancer chemotherapy.

ErbB2 (HER-2) gene amplification and overexpression have been shown to predict a better outcome with doxorubicin-based chemotherapy as opposed to alkylator-based chemotherapy in early stage breast cancer. To understand the mechanism of differential response to these two regimens, we have evaluated the effect of signaling through the ErbB2 receptor on downstream enzymes that may affect drug response, using two different models. The first system employs breast cancer cells that have high levels of endogenous ErbB2 by gene amplification (BT-474 and SKBR3 cells). The second system allows us to isolate the effect of ErbB2 receptor-mediated intracellular signaling using an epidermal growth factor receptor-ErbB2 chimeric receptor activated by epidermal growth factor. Our experiments show that the cytotoxicity of doxorubicin is inhibited in ErbB2+ breast cancer cells by the anti-ErbB2 antibody, Herceptin. This is accompanied by a decrease in topoisomerase (topo) IIalpha protein and activity, suggesting that this is the mechanism of change in doxorubicin response. In addition, a 10-100-fold (1-2 log) decrease in the LD(50) of doxorubicin is seen after ErbB2 activation using the chimeric receptor model. Furthermore, we see a 100-fold decrease in the LD(50) of etoposide, another topo II inhibitor. This increase in doxorubicin sensitivity is associated with a 4.5-fold increase in the amount of topo IIalpha protein and an increase in topo II activity as measured by DNA decatenating and unknotting activities, as well as cleavable complex formation. In contradistinction to doxorubicin, we have observed an increased resistance to cyclophosphamide chemotherapy after chimeric receptor activation. We propose that the differential benefit seen with doxorubicin- versus alkylator-based chemotherapy in ErbB2+ breast cancer is due, in some cases, to ErbB2-mediated topo IIalpha activation. These data also suggest hypotheses for the optimal sequencing of Herceptin and chemotherapy agents in ErbB2+ breast cancer.

Atp-bound topoisomerase ii as a target for antitumor drugs.

Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.

Essential functions of DNA topoisomerase I in Drosophila melanogaster.

DNA topoisomerase I (topo I) is an essential enzyme involved in replication, transcription, and recombination. To probe the functions of topo I during Drosophila development, we used top1-deficient flies with heat-shock-inducible top1 transgenes and were able to observe both zygotic and maternal functions of top1. A critical period for the zygotic function is in the late larval and early pupal stages. Topo I is required for larval growth and cell proliferation in imaginal disc tissues. The maternal functions consist of two aspects: oogenesis and early embryogenesis. During oogenesis, topo I is detected in the nuclei of early germ-line cells and follicle cells. The mutant ovary exhibits abnormal proliferation and defective nuclear morphology in these cells. There are extranumeral germ-line cells in individual egg chambers, while the follicle cells are underreplicated. Topo I is also stored maternally in early embryos. It localizes to the nuclei during interphase and prophase, but disperses into the cytoplasm at metaphase. Embryos from the mutant mother frequently show disrupted nuclear divisions with defects in chromosome condensation and segregation. The cytological and genetic analysis of the top1 mutant demonstrates that in Drosophila, topo I plays critical roles in many developmental stages active in cell proliferation.

Discontinuous actin hexagon, a protein essential for cortical furrow formation in Drosophila, is membrane associated and hyperphosphorylated.

discontinuous actin hexagon (dah) is a maternal-effect gene essential for the formation of cortical furrows during Drosophila embryogenesis, and DAH protein colocalizes with actin in these furrows. Biochemical fractionation experiments presented here demonstrate that DAH is highly enriched in the membrane fraction and that its membrane association is resistant to high-salt and alkaline washes. Furthermore, it partitions into the detergent phase of the Triton X-114 solution, indicating its tight binding to the membranes. DAH can also interact with the actin cytoskeleton, because a fraction of DAH remains insoluble to nonionic detergent along with actin. These biochemical characterizations suggest that DAH may play a role in the linkage of the actin cytoskeleton to membranes. Using phosphatase inhibitors, we detected multiple phosphorylated forms of DAH in embryonic extracts. The DAH phosphorylation peaks during cellularization, a stage at which DAH function is critical. A kinase activity is coimmunoprecipitated with the DAH complex and hyperphosphorylates DAH in vitro. Purified casein kinase I can also hyperphosphorylate DAH in the immune complex. Both DAH localization and phosphorylation are disrupted in another maternal-effect mutant, nuclear-fallout. It is possible that nuclear-fallout collaborates with dah and directs DAH protein localization to the cortical furrows.

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.

The Drosophila centrosomal protein Nuf is required for recruiting Dah, a membrane associated protein, to furrows in the early embryo.

During mitosis of the Drosophila cortical syncytial divisions, actin-based membrane furrows separate adjacent spindles. Our genetic analysis indicates that the centrosomal protein Nuf is specifically required for recruitment of components to the furrows and the membrane-associated protein Dah is primarily required for the inward invagination of the furrow membrane. Recruitment of actin, anillin and peanut to the furrows occurs normally in dah-derived embryos. However, subsequent invagination of the furrows fails in dah-derived embryos and the septins become dispersed throughout the cytoplasm. This indicates that stable septin localization requires Dah-mediated furrow invagination. Close examination of actin and Dah localization in wild-type embryos reveals that they associate in adjacent particles during interphase and co-localize in the invaginating furrows during prophase and metaphase. We show that the Nuf centrosomal protein is required for recruiting the membrane-associated protein Dah to the furrows. In nuf-mutant embryos, much of the Dah does not reach the furrows and remains in a punctate distribution. This suggests that Dah is recruited to the furrows in vesicles and that the recruiting step is disrupted in nuf mutants. These studies lead to a model in which the centrosomes play an important role in the transport of membrane-associated proteins and other components to the developing furrows.

The hydrophilic, protease-sensitive terminal domains of eucaryotic DNA topoisomerases have essential intracellular functions.

The amino-terminus of eucaryotic DNA topoisomerase I and the carboxy-terminus of eucaryotic DNA topoisomerase II contain sequences that are enriched in charged amino acid residues, hyper-sensitive to protease digestion, not required for the in vitro topoisomerase activities, able to tolerate insertion and deletion mutations, and thus may have a disordered structure. In an interesting contrast to the catalytically essential core domain, the sequences in these terminal hydrophilic domains are not conserved among the topoisomerases from different species. However, many lines of evidence, including those presented here, demonstrate that the topoisomerase tail domains have critical intracellular functions. The biological functions of the amino-terminus of topoisomerase I include the nuclear import and targeting to the transcriptionally active loci. The carboxy-terminus of topoisomerase II also contains the sequences necessary for nuclear localization and possibly sequences necessary for other critical functions.

Penile cancer in Taiwan--20 years' experience at National Taiwan University Hospital.

To analyze the characteristics and prognostic factors of penile cancer in Taiwanese, we retrospectively reviewed the clinical data of patients with a diagnosis of penile cancer treated during a 20-year period (1977-1996) at National Taiwan University Hospital (NTUH). Of 71 patients treated for penile cancer during the study period, 17 were referred from other hospitals or clinics. Our analyses focused on the 54 previously untreated patients. Growth on the penis was the main symptom in all cases. Palpable inguinal lymph nodes were found only in 14 patients. All 54 patients with primary tumors were treated surgically. Pathologic examination showed squamous cell carcinoma (SCC) in 43 cases, extra-mammary Paget's disease in three, verrucous carcinoma in three, Bowen's disease in two, cutaneous lymphoma in two and basal cell carcinoma in one. Twenty-six (48%) patients had stage I penile cancer, 13 (24%) had stage II, seven (13%) had stage III, and eight (15%) had stage IV cancer. The five-year survival rate was 78% among patients with SCC and 84% among those with nonsquamous malignancies (p = 0.80). The five-year cumulative survival rates according to Jackson's cancer stage were 100% for patients with stage I, 88.9% for those with stage II, 66.7% for those with stage III, and 0% for those with stage IV (p < 0.001). Tumor staging (p = 0.027) and adjuvant chemotherapy (p = 0.042) were found to be the most significant prognostic factors. Penile cancer accounted for 0.254% of all malignancies among male patients at the NTUH during the study period. Our findings indicate that penile cancer is uncommon in Taiwanese and its prognosis is closely related to tumor staging and management. Early diagnosis and appropriate treatment may lead to prolonged survival.

Complex response of breast epithelial cell lines to topoisomerase inhibitors.

The topoisomerase inhibitors, camptothecin and etoposide target the activity of topoisomerase I and II respectively. These agents, or their analogues, are undergoing clinical trials for the treatment of metastatic breast cancer. In this study, we examined the response of eight breast epithelial cell lines, including six lines derived from breast cancers and two immortalized normal epithelial lines to camptothecin and etoposide. The lines varied by 700 fold in their sensitivity to the growth inhibiting effects of camptothecin and 30 fold in their response to etoposide. The BT474 line was the most resistant to both agents. The other cell lines did not have uniform sensitivity to both drugs, i.e., some lines were sensitive to one drug but relatively resistant to the other. A variety of parameters in these lines were analyzed to elucidate mechanisms of resistance including S phase, doubling time, expression and activity of topoisomerase I and II, expression of mdr-1, p53 status, cell cycle arrest, level of apoptosis, and expression of the apoptotic proteins Bcl-2 and Bax. We found that low levels of the topo I protein and its enzymatic activity were associated with increased resistance to camptothecin. This was not true for topo II activity and etoposide. Increased apoptotic responses were generally observed in cell lines that were sensitive to etoposide and this correlated with low ratios of Bcl-2/Bax protein. No single parameter was entirely predictive of response. However, the BT474 line displayed a series of characteristics including slow growth, the presence of mutant p53, low topo I activity, and a high Bcl-2/Bax ratio which together likely contributed to the resistance of this line to both etoposide and camptothecin.

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159.

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.

Structure of the Drosophila DNA topoisomerase I gene and expression of messages with different lengths in the 3' untranslated region.

The nucleotide sequence of the Drosophila DNA topoisomerase I gene (top1) has been determined. Structurally, top1 consists of eight exons and seven introns. The top1 coding region contains a new class of opa repeats, encoding clusters of serine residues instead of glutamine repeats usually seen in Drosophila genes of the neurogenic loci. A unique feature of top1 is the developmental switch of its transcripts: a heterogeneous population of transcripts ranging from 3.8 to 4.2kb seen maximally at 0-2h of embryogenesis and a 5.2-kb transcript maximal at 6-12h of embryonic development. The transcripts expressed in the 0-2-h embryo have been shown as maternal storage products specific to ovarian tissues. RACE analysis shows that whereas the 6-12-h transcripts have a single site for polyadenylation, there are at least 12 different sites for poly(A) addition to the 0-2-h transcripts. An additional intron specific for the maternal storage transcripts appears in some of the 0-2-h transcripts. No significant heterogeneity at the 5' end of the top1 transcripts is seen. Sequence searches have revealed a number of regulatory sequences for potential translational control in the 3' untranslated region.

Targeting to transcriptionally active loci by the hydrophilic N-terminal domain of Drosophila DNA topoisomerase I.

DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against beta-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.

Combined cytotoxic effects of tamoxifen and chemotherapeutic agents on bladder cancer cells: a potential use in intravesical chemotherapy.

OBJECTIVE: To evaluate whether tamoxifen enhances the cytotoxicity of chemotherapeutic agents on bladder cancer cells, and the possible mechanism(s) of action. MATERIALS AND METHODS: The in vitro inhibition of cell growth was examined in a model simulating intravesical chemotherapy using two bladder cancer cell lines (TSGH-8301, HTB9) and three commonly used intravesical cytotoxic agents (doxorubicin, mitomycin C, and thiotepa) in the presence or absence of tamoxifen or verapamil as modulators. The expression of the multi-drug resistance-related gene mdr-1 was evaluated by reverse-transcription polymerase chain reaction and Southern blotting (RT-PCR-SB) to determine its transcript level, by flow cytometric analysis of the P-glycoprotein (P-gp) product level with C-219 monoclonal antibody and by the rhodamine-123 retention and efflux assay for P-gp activity. Transforming growth factor beta-1 (TGF beta-1) levels in tamoxifen-conditioned culture medium were determined with enzyme-linked immunosorbant assay (ELISA). RESULTS: Tamoxifen at concentrations > or = 30 microM significantly enhanced the cytotoxicity of the three chemotherapeutic agents to both cell lines, as shown by a marked reduction in the drug concentration which inhibited growth by 50% (IC50). The enhancement of cytotoxicity was significantly dependent on the concentration of tamoxifen. However, tamoxifen alone caused significant toxic effects to TSGH-8301 at > or = 40 microM and to HTB9 at > or = 30 microM. Median-effect analysis showed additive or less-than-additive combination effects between tamoxifen and chemotherapeutic agents and only a minimal synergism in a narrow range of maximal cytotoxicity (fraction affected > 0.9). Thus, the reduction of IC50s by tamoxifen was mostly because it was cytotoxic to the bladder cancer cells used. No enhancement of cytotoxicity was observed in verapamil-modulated cells. Transcripts of mdr-1 could not be detected by RT-PCR-SB, nor was P-gp detected by flow cytometric analysis in the two cell lines. Furthermore, no active P-gp function was detected by the rhodamine-123 retention and efflux study, indicating that the primary chemoresistance mechanisms of the two cell lines were not mediated by mdr-1, nor could tamoxifen or verapamil act through modulation of the mdr-1 pathway in the two cell lines. Tamoxifen at 3 and 10 microM down-regulated the secretion of TGF beta-1 from TSGH-8301 in a concentration-dependent manner, in contrast to the findings that tamoxifen was cytotoxic to the bladder cancer cells used and that tamoxifen up-regulated TGF beta-1 in a breast cancer model, suggesting that there may be a different mechanism of response to TGF beta-1 in these bladder cancer cells. CONCLUSION: Tamoxifen enhanced the cytotoxicity of chemotherapeutic agents largely through its toxic effects on the bladder cancer cells. The mode of action of tamoxifen was not through the regulation of TGF beta-1 or the function of mdr-1. Although cytotoxic levels of tamoxifen (> 50 microM) can be achieved easily in the intravesical model, further study is necessary before tamoxifen can be used clinically in intravesical chemotherapy.

Tamoxifen enhances the chemosensitivity of bladder carcinoma cells.

PURPOSE: Chemosensitizers, which enhance cellular chemosensitivity and reduce chemoresistance, are expected to substantially improve response rates of systemic chemotherapy for patients with metastatic bladder cancer. Tamoxifen is a nonsteroidal antiestrogen that has been shown to reverse drug resistance in vitro in some cancer models through pathways not related to its antiestrogenic effect. In this study we tried to evaluate its possible effect on chemosensitization of bladder cancer cells. MATERIALS AND METHODS: In vitro chemosensitivity tests were done with 3 bladder cancer cell lines and 4 cytotoxic agents (methotrexate, vinblastine, doxorubicin and cisplatin) in the presence or absence of graded concentrations of tamoxifen. Verapamil was used in parallel experiments to compare the degrees of chemosensitization. The transcript and protein product [P-glycoprotein, (P-gp)] levels of the mdr-1 gene were also examined in the 3 cell lines by reverse-transcription polymerase chain reaction (RT-PCR) and flow cytometric assay, respectively. RESULTS: Tamoxifen at 5 and especially 10 microM. concentrations, which were minimally toxic to the 3 bladder cancer cell lines used, enhanced the chemosensitivity of bladder cancer cells in most drug combinations in a dose-dependent manner. In some combinations 10 microM. tamoxifen did better than 5 microM. in chemosensitization. The effect of chemosensitization was more evident in cells treated with 10 microM. tamoxifen plus methotrexate and vinblastine in which 12.9 to 95.4-fold and 12.4 to 21.3-fold IC50 reduction was observed, respectively. A less prominent, but still significant, effect could be seen in doxorubicin- and cisplatin-treated cells. Verapamil, although used at concentrations up to 10 microM. which are higher than systemically tolerable, was not able to enhance chemosensitivity of the 3 bladder cancer cell lines. By flow cytometric analysis of the P-gp level and by RT-PCR assay of the mdr-1 gene transcript level, it was shown that little if any mdr-1 gene expression could be detected in the 3 cell lines. This implies that the mdr-1 gene function may play a minimal role in drug resistance mechanisms of bladder cancer cells and that tamoxifen exerts its chemosensitization effect through pathways other than mdr-1 gene function modulation. CONCLUSIONS: Tamoxifen was shown to be a good chemosensitizer in a bladder cancer cell model and may well be tried in combination with systemic chemotherapy for metastatic human bladder cancers in the clinical setting.

Expressions of E-cadherin and exon v6-containing isoforms of CD44 and their prognostic values in human transitional cell carcinoma.

Cell surface adhesion molecules, E-cadherin and exon v6 containing CD44 isoforms (CD44v6), were readily found in well-to-moderately differentiated urothelial cell lines but were down-regulated in poorly differentiated cell lines. One hundred and fifteen tumors of transitional cell carcinoma (TCC) were examined with E-cadherin and CD44v6 specific antibodies. Sixty-five (56.5%) tumors exhibited a preserved type while 50 (43.5%) showed a reduced type for CD44v6. Sixty-seven (58.3%) tumors were classified as the preserved type, and 48 (41.7%) were classified as the reduced type for E-cadherin. The staining pattern of E-cadherin was the same as that of CD44v6 in 87.0% (100 of 115) of tumors. The frequency of the reduced type was higher in poorly differentiated carcinomas (32 of 52 for CD44v6, p = 0.001; 27 of 52 for E-cadherin, p = 0.112) and tumors with an invasive growth pattern (22 of 27 for CD44v6, p < 0.001; 20 of 27 for E-cadherin, p < 0.001) than it was in well-differentiated carcinomas and tumors with expansile growth. However, the association with lymph node involvement or distant metastasis did not reach statistical significance. There was no difference in survival in reference to the expression patterns of CD44v6 and E-cadherin. Furthermore, neither marker was a significant prognostic factor for tumor recurrence and survival according to Cox's multiple variant regression analysis.

Zona fasciculata-like cells determine the response of plasma aldosterone to metoclopramide and aldosterone synthase messenger ribonucleic acid level in aldosterone-producing adenoma.

The different responses of plasma aldosterone to ACTH and angiotensin II in aldosterone-producing adenoma (APA) is thought to be due to the various cellular compositions of the tumors. To investigate whether the dopaminergic regulation of aldosterone in APA is also dependent on the cellular types, we studied the effects of metoclopramide on plasma aldosterone in six patients with APA. The messenger RNA (mRNA) levels of aldosterone synthase (P450aldo), 11 beta-hydroxylase (P450(11) beta), and 17 alpha-hydroxylase (P450(17) alpha) of APA and normal adrenal glands were determined by competitive polymerase chain reaction. After administration of metoclopramide (an antagonist of dopamine-2 receptor), the increment of plasma aldosterone correlated inversely with the percentage of zona fasciculata cells of APA. The mRNA level of P450aldo in the tumorous portion was much higher, whereas the levels of P450(11) beta and P450(17) alpha mRNAs were lower, than those of the nontumorous portion and normal adrenals. There was a correlation of the percentage of zona fasciculata cells in APA with the levels of P450aldo and P450(11) beta mRNAs, but not with P450(17) alpha mRNA. These results suggest that differential responsiveness of plasma aldosterone to metoclopramide may be due to various proportions of different cell types in APA that may have different expression of dopamine-2 receptor. In addition, this histologically dependent expression was present at the transcriptional level of the gene responsible for aldosterone biosynthesis.

Ileal neobladder reconstruction after radical cystoprostatectomy.

To improve the outcome and patient acceptance of bladder substitution, five male patients underwent O-shaped ileal neobladder reconstruction, after radical cystoprostatectomy for invasive bladder cancer, with a mean follow-up of 11 months. With only 40 cm of ileal segment, an O-shaped neobladder was constructed after complete detubularization. Bilateral ureters were implanted using the Le Duc-Camey method. Six months after operation, all patients were totally continent during the day time, and one patient suffered from mild incontinence at night, which could be overcome by waking to void once or twice. The satisfactory continence levels are in agreement with a urodynamic study of the neobladder which showed a low pressure, high-capacity (mean, 456 mL) reservoir in the cystometric tracings. The mean maximal flow rate was 22.2 mL/sec, the mean residual urine was minimal (10 to 20 mL), the mean maximal urethral closing pressure was 74.4 cm H2O and the mean functional profile length was 2.9 cm. All renal units do not have neovesico-ureteral reflux. Two patients showed unilateral hydronephrosis which subsided later, one patient sustained bilateral hydronephrosis and died of jejunal perforation five months postoperatively. There were few perioperative complications and no patient expressed regret at having undergone the procedure. We consider bladder substitution to be the treatment of choice in male patients requiring radical cystoprostatectomy.

Role of urinary cytology and urinary deoxyribonucleic acid flow cytometry in the diagnosis of bladder cancer.

In order to determine the diagnostic efficiencies of urinary cytology and urinary deoxyribonucleic acid (DNA) flow cytometry (FCM) in the detection of bladder cancer, 92 patients were studied from March 1991 to the end of July 1992. Thirty cases had previously undergone operation for bladder cancer and 62 cases were suspected as bladder cancer. One to three fresh voided urine samples from the same patient were sent for conventional urinary cytology and urinary DNA FCM analysis. Each patient underwent cystoscopic examination or surgical histopathologic examination to verify the presence of bladder tumor. From December 1991 to the end of July 1992, 52 cases were analyzed and reported separately due to improved FCM techniques. Urinary DNA FCM showed a higher sensitivity than cytology in both the total (p < 0.05) and the second half time period (p < 0.01). Cytology showed a statistically superior specificity against FCM in the total period (p < 0.05) but not in the second half time period (p > 0.1). Combining sensitivity and specificity, FCM's overall accuracy rate was better than cytology in the second half time period (p < 0.05). To clarify the specific features of bladder tumors in which FCM showed superior sensitivity than cytology, we analysed the detection rates for various features of bladder tumor in the second half time period. FCM was better than cytology in detecting multiple tumors, small tumors and papillary tumors. No statistical differences were obtained if tumors were single, larger than 3 cm or flat in outer surface. To our knowledge, no previous similar analysis has been reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)

Linker insertion mutagenesis of Drosophila topoisomerase II. Probing the structure of eukaryotic topoisomerase II.

The sequences of all type II DNA topoisomerases, and possibly some of their key structural features, are conserved. The N-terminal and middle regions of the eukaryotic DNA topoisomerase II are homologous to the bacterial gyrase subunits B and A, respectively, and the hydrophilic C-terminal region is more divergent among these enzymes. To gain further insights into the structure of eukaryotic topoisomerase II, we constructed 23 linker insertion mutants of Drosophila DNA topoisomerase II. These mutant proteins were expressed in a heterologous yeast system, in which we have previously demonstrated that Drosophila DNA topoisomerase II could be functionally expressed and complement yeast top2 mutations. The linker insertion mutants were characterized genetically by testing for complementation of yeast top2ts mutation at the non-permissive temperature and complementation of yeast top2 null mutation using a color sector assay. We also partially purified the mutant proteins and examined their enzymatic activity by unknotting the P4 knotted DNA. There appears to be a good correlation between the in vivo and in vitro activities. There are nine fully active, six partially active, and eight negative linker insertion mutants. All five linker insertion mutants in the C-terminal region are active and two linker insertion mutants located in the junction of the two regions homologous to gyrB/gyrA subunits are also active. In addition, we also mapped the trypsin-sensitive sites in Drosophila DNA topoisomerase II. The C-terminal region is extremely sensitive to trypsin digestion. Another major trypsin-sensitive site is located between Lys406 and Thr407, which is near the protease sites also observed in the bacterial gyrB subunit and yeast topoisomerase II. We discuss the possible structural and functional implications of these results.