DNA topoisomerases.

DNA topoisomerases play an important role in regulating DNA structure, thus affecting many aspects of chromosome function inside cells. Recent progress in this field raises exciting questions regarding the distinct and critical functions of multiple topoisomerases, and the roles of DNA topoisomerases in the processes of chromosome condensation, decondensation, and segregation.

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.

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.

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.

Topoisomerase II cleavage in chromatin.

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.

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.

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.

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.

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.

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.

Characterization of a newly established human bladder carcinoma cell line, NTUB1.

A new human bladder cancer cell line, NTUB1, has been derived from the surgical specimen of a 70-year-old female patient diagnosed with poorly differentiated transitional cell carcinoma. It has been successfully propagated in vitro for over 24 months without evidence of reaching senescence. Population doubling time was about 21 hours at the 32nd passage. It was tumorigenic in nude mice, and the histologic findings of the heterotransplanted tumor resembled the original tumor. Expression of keratin proteins confirmed its epithelial origin. Cytogenetic analysis showed multiple chromosome changes. Anticancer drugs, including thiotepa and adriamycin, were tested in vitro, and the cytotoxicity did not exceed 50% of the control value; likewise, in this patient chemotherapy was not effective. On the other hand, a combination of recombinant tumor necrosis factor and interferon tau in vitro was more effective against this tumor.

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)

Serum level of immunosuppressive acidic protein in patients with urological malignancies.

Immunosuppressive acidic protein (IAP) is a non-specific immunoreactive protein arising from inflammatory or malignant conditions in the human body. We determined the IAP levels in 65 cases with urological malignancies and in 31 cases with benign diseases as a control group during a 9-month period. There were significantly higher serum levels of IAP in cases of bladder transitional cell carcinoma (p = 0.025), prostate adenocarcinoma (p less than 0.00001) and upper urinary tract urothelial cancer (kidney and/or ureter, p = 0.013) as compared with those of the control group. Significant differences in IAP between different tumor stages were found in the bladder cancer group with high stage cases having higher IAP levels (p less than 0.0005). However, no significant differences were found between different tumor gradings. Most of the prostate cancer patients had extremely high IAP values (1,029 +/- 490 micrograms/ml) in this study. Renal cell carcinoma and testicular tumors showed no statistical differences from the control group (p = 0.89 and 0.37, respectively). No differences could be found in the different age groups (by decades) or sexes. The serum IAP level can be a good non-specific tumor marker for bladder cancer staging and probably a good follow-up tool for most urological malignancy patients.

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.

[Clinical analysis of urolithiasis in Poh Ai Hospital of I-Lan, Taiwan, R.O.C.--a comparative study with urolithiasis in Japan].

Between August 1987 and December 1990, 546 patients were admitted to the department of Urology at the Poh Ai Hospital of I-Lan, Taiwan, R.O.C. for the treatment of urinary stones. These urinary stone cases accounted for 50 to 60% of all urology patients admitted. The incidence of urolithiasis in I-Lan was estimated at 147/100,000 population in 1990. There were 402 male patients and 144 female patients, The male to female ratio was 2.8: 1. There were 450 upper urinary tract stones (kidney, ureter) in 314 males and 136 females, and 79 lower urinary tract stones (bladder, urethra) in 72 males and 7 females. The ratio of upper to lower urinary tract stones was 6:1. Endourological treatments such as percutaneous nephrolithotripsy and transurethral ureterolithotripsy have increased rapidly in recent years. A summary of the present analysis for composition of 365 stones follows. The most frequent type was calcium-containing stone (92.3%), followed by infection stone (4.7%), then uric acid (UA) stone (3.0%). There were no UA stones found in the female patients. According to urinalysis criteria of more than 10 WBC/HPF (x 400), pyuria was found in 67 cases of 334 metabolic stones (20.1%), and 11 cases of 17 infection stones (67.7%). There were neither pediatric case of stone formation nor cystine stones.

Functional expression of a Drosophila gene in yeast: genetic complementation of DNA topoisomerase II.

Since DNA topoisomerase II (EC 5.99.1.3) is an essential enzyme in yeast, heterologous topoisomerase II gene expression in yeast cells can provide a system for analyzing the structure and function of topoisomerase II genes from other species. A series of yeast expression plasmids was constructed in which segments of the cDNA sequences encoding Drosophila DNA topoisomerase II were inserted under the transcriptional control of yeast GAL1 promoter. Expression of the functional form of Drosophila topoisomerase II cDNA can complement conditionally lethal, temperature-sensitive mutations in the yeast topoisomerase II gene (TOP2), as well as mutations in which the TOP2 locus was disrupted. The survival of these yeast cells depends upon the continuous expression of Drosophila topoisomerase II. Repression of Drosophila gene expression by glucose causes these yeast cells to cease dividing after a few generations. In addition to these genetic complementation data, the expression of the Drosophila topoisomerase II gene in yeast cells with a disruption in TOP2 can also be detected by immunochemical methods with an antibody specific for Drosophila topoisomerase II.

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.