Topo IIalpha Research Articles

Expression of glutathione S-transferase-pi and DNA topoisomerase II alpha and their implications in colorectal carcinoma

To investigate the expression of GST-pi and Topo II-alpha, and their relationships with clinicopathological parameters in colorectal carcinoma. The expression of GST-pi and Topo II-alpha were detected by avidin-biotin-peroxide complex (ABC) method in tumor specimens, matched paratumor tissues from 60 cases with colorectal carcinoma and normal colonic tissues from 15 cases. The expression rates of GST-pi and Topo II-alpha were 90.0% and 86.7% respectively in tumor tissues, significantly higher than those in matched paratumor tissues and normal tissues (P< 0.01). The expressions of GST-pi and Topo II-alpha were associated with cellular differentiation, Dukes stage and lymph node metastasis (all P< 0.01), but not with tumor size and histological type (all P > 0.05). The expression level of GST-pi was significantly higher in poorly differentiated tumors than that in well differentiated tumors. The expression level of Topo II-alpha in well-differentiated tumors were stronger than that in poorly differentiated tumors. The detection of GST-pi and Topo II-alpha expressions may be helpful to judge the malignant behavior, metastasis and prognosis in human colorectal carcinoma.

Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIα for anthracycline chemotherapy in locally advanced breast cancer

Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIalpha (topo IIalpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIalpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. Thirty-three women with primary breast tumors > or =3 cm received either doxorubicin (75 mg/m(2)) or epirubicin (120 mg/m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24-48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIalpha at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIalpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

Altered expression of topoisomerase IIα contributes to cross-resistant to etoposide K562/MX2 cell line by aberrant methylation

KRN 8602 (MX2) is a novel morpholino anthracycline derivative having the chemical structure 3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin hydrochloride. To investigate the mechanisms of resistance to MX2, we established an MX2-resistant phenotype (K562/MX2) of the human myelogeneous leukaemia cell line (K562/P), by continuously exposing a suspension culture to increasing concentrations of MX2. K562/MX2 cells were more resistant to MX2 than the parent cells, and also showed cross-resistance to etoposide and doxorubicin. Topoisomerase (Topo) IIα protein levels in K562/MX2 cells were lower of those in K562/P cells on immunoblot analysis and decreased expression of Topo IIα mRNA was seen in K562/MX2 cells. Topoisomerase II catalytic activity was also reduced in the nuclear extracts from K562/MX2 cells when compared with K562/P cells. Aberrant methylated CpG of Topo IIα gene was observed in K562/MX2 cells when compared with the parent line on methylation-specific restriction enzyme analysis. To overcome the drug resistance to MX2 and etoposide, we investigated treatment with 5-Aza-2′-deoxycytidine (5AZ), which is a demethylating agent, in K562/MX2 cells. 5-Aza-2′-deoxycytidine treatment increased Topo IIα mRNA expression in K562/MX2 cells, but not in K562/P cells, and increased the cytotoxicity of MX2 and etoposide. Methylated CpG was decreased in K562/MX2 cells after 5AZ treatment. We concluded that the mechanism of drug resistance to MX2 and etoposide in K562/MX2 cells might be the combination of decreased expression of Topo IIα gene and increased methylation, and that 5AZ could prove to be a novel treatment for etoposide-resistant cell lines, such as K562/MX2.

Phenotypic characterization of BRCA1 and BRCA2 tumors based in a tissue microarray study with 37 immunohistochemical markers

Familial breast cancers that are associated with BRCA1 or BRCA2 germline mutations differ in both their morphological and immunohistochemical characteristics. To further characterize the molecular difference between genotypes, the authors evaluated the expression of 37 immunohistochemical markers in a tissue microarray (TMA) containing cores from 20 BRCA1, 14 BRCA2, and 59 sporadic age-matched breast carcinomas. Markers analyzed included, amog others, common markers in breast cancer, such as hormone receptors, p53 and HER2, along with 15 molecules involved in cell cycle regulation, such as cyclins, cyclin dependent kinases (CDK) and CDK inhibitors (CDKI), apoptosis markers, such as BCL2 and active caspase 3, and two basal/myoepithelial markers (CK 5/6 and P-cadherin). In addition, we analyzed the amplification of CCND1, CCNE, HER2 and MYC by FISH. Unsupervised cluster data analysis of both hereditary and sporadic cases using the complete set of immunohistochemical markers demonstrated that most BRCA1-associated carcinomas grouped in a branch of ER-, HER2-negative tumors that expressed basal cell markers and/or p53 and had higher expression of activated caspase 3. The cell cycle proteins associated with these tumors were E2F6, cyclins A, B1 and E, SKP2 and Topo IIalpha. In contrast, most BRCA2-associated carcinomas grouped in a branch composed by ER/PR/BCL2-positive tumors with a higher expression of the cell cycle proteins cyclin D1, cyclin D3, p27, p16, p21, CDK4, CDK2 and CDK1. In conclusion, our study in hereditary breast cancer tumors analyzing 37 immunohistochemical markers, define the molecular differences between BRCA1 and BRCA2 tumors with respect to hormonal receptors, cell cycle, apoptosis and basal cell markers.

In Vitro Effects of the Combination of Idarubicin (IDA) with Suberoylanilide Hydroxamic Acid (SAHA) or Valproic Acid (VPA) in Leukemia Cell Lines.

The nucleosome is the basic structure of chromatin. Changes in the biochemical composition of nucleosome-associated histone tails are associated with specific gene activation states, and are the target of several antineoplastic agents such as histone deacetylase inhibitors (HDI). Nucleosomes are constrained into loops that are flanked by domains known as matrix-attached regions (MARs). MARs contain DNA topoisomerase II (Topo II) consensus sequences. Topo II is responsible for regulating and maintaining DNA topology and is the target of several antineoplastic agents such as the anthracycline IDA, an effect mediated by the induction of double strand DNA breaks (DSB). We hypothesized that the combination of a Topo II inhibitor and a HDI will have synergistic antileukemia activity. VPA and SAHA are two HDIs currently studied in several clinical trials with known antileukemia activity and tolerable toxicity. To test our hypothesis and to develop future clinical studies, we have analyzed the effect of the combination of IDA, a potent Topo II inhibitor, with VPA or SAHA. We treated the leukemic cells lines MOLT4 and HL60 with increasing doses of IDA (0.5-20nM), SAHA (0.3-3μM) or VPA (0.25-3mM) daily for 3 days. First, using trypan blue viability assays, we identified the IC10 of IDA to be 0.5nM for MOLT4 and 1.5nM for HL60. Doses in excess of 2μM of SAHA or 3mM of VPA resulted in more than 90% decrease in cell viability in both cell lines. Subsequently, SAHA at doses of 0.075-1μM and VPA at 1-3mM were used for the combination experiments with IDA at its specific cell line IC10. At low doses of SAHA (0.075-0.45 μM) and VPA (0.25-1 mM) the combination was shown to have synergistic antileukemia activity by the Fractional Product Method of Webb. These results were confirmed using Annexin V assays. Of importance, growth inhibition was independent of the sequence used. To analyze the effects of this combination on DSB generation, we analyzed using immonocytochemistry and western blot, the induction of γH2AX, a histone variant that has been identified as an early event after the DSBs. SAHA alone induced a modest increase in γH2AX compared to baseline, whereas IDA alone had a significant effect that was not potentiated by the addition of SAHA. Histone H3 and H4 acetylation increased in a dose-dependent manner (2.4–15 fold) with both SAHA and VPA, starting at 0.3μM of SAHA and 0.25mM of VPA. The addition of IDA had no significant effect on histone acetylation. Because of previous data indicating that HDIs may down-regulate the expression of Topo II-alpha, the target of IDA, we have studied using real-time PCR its levels prior and during exposure to the different combinations. SAHA or VPA had no effect on Topo II-alpha mRNA levels whereas IDA induced 2.0–3.5 fold its expression in a dose-independent manner, an effect no altered by the addition of SAHA or VPA. Expression of p21CIP1, that is silenced in both cell lines, was restored by single agent VPA, SAHA or IDA. The combination of these drugs resulted in an additive effect in terms of p21CIP1 induction. Despite this phenomenon, no changes in cell cycle status were observed in these cells. In summary, the combination of IDA and SAHA or VPA has potent in vitro antileukemia effect, and should be studied in clinical trials.

Prognostic value of Topoisomerase II in female breast cancer

Topoisomerase II-alpha (Topo II-alpha) is a nuclear enzyme. Its expression rises rapidly at the end of the S to G2/M phase and falls after the mitotic process ends. We have studied the immunohistochemical expression of Topo II-alpha in breast cancer and its correlation with the menopausal state, tumor type, size, lymph node metastases, stage, and estrogen and progesterone positivity. Histological sections from 50 breast cancers were immunohistochemically stained for Topo II-alpha. The percent of positive cells at the area of highest staining was recorded as Topo index. The correlation between the course of disease, survival and Topo II-alpha index was statistically significant, p<0.001. High-grade tumors showed higher Topo II-alpha levels, than those of intermediate and low-grade, p<0.01. A significant association was found between estrogen receptors positivity and Topo II-alpha, p<0.05. A higher Topo II-alpha index indicates higher probability for recurrence of the disease and overall survival. Therefore, Topo II-alpha expression has a prognostic value in breast carcinoma.

Effects of Scaffold/Matrix Alteration on Centromeric Function and Gene Expression

We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.

Interaction between Glucose-regulated Destruction Domain of DNA Topoisomerase IIα and MPN Domain of Jab1/CSN5

DNA topoisomerase (topo) IIalpha, an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glucose-starved. Here we show that the topo IIalpha destabilization depends on the newly identified domain, GRDD (glucose-regulated destruction domain), which was mapped to the N-terminal 70-170 amino acid sequence. Indeed, the deletion of GRDD conferred a stable feature on topo IIalpha, whereas the fusion of GRDD rendered green fluorescent protein unstable under glucose starvation conditions. Nuclear localization was a prerequisite for GRDD function, because the inhibition of nuclear translocation resulted in the suppression of GRDD-mediated topo IIalpha degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo IIalpha in a manner dependent on the MPN (Mpr1p/Prd1p N terminus) domain. Depleting Jab1/CSN5 by antisense oligonucleotide and treating cells with the CSN-associated kinase inhibitor, curcumin, inhibited topo IIalpha degradation induced by glucose starvation. These findings demonstrate that GRDD can act as a stress-activated degron for regulating topo IIalpha stability, possibly through interaction with the MPN domain of Jab1/CSN5.

Topoisomerase IIalpha expression in mantle cell lymphoma: a marker of cell proliferation and a prognostic factor for clinical outcome.

Mantle cell lymphoma (MCL) is a malignant lymphoma associated with a relatively aggressive clinical course and a median overall survival time of 3-4 years. Treatment usually consists of combination chemotherapy, often including topoisomerase (topo) inhibitors such as doxorubicin, etoposide and mitoxantrone. Topo IIalpha is an enzyme that is needed whenever uncoiling of DNA is necessary during the cell cycle. The enzyme is a marker of cell proliferation. We analyzed the expression of topo IIalpha in relation to Ki-67 and the clinical outcome in patients with MCL. Biopsy specimens from 95 untreated patients enrolled in two multicenter trials (1975-1985) were investigated immunohistochemically with monoclonal antibodies against topo IIalpha (Ki-S4) and Ki-67 (Ki-S5). Patients with low (0-10%) topo IIalpha expression had a median overall survival time of 49.0 months, compared to 17.0 months for patients with high (more than 10%) topo IIalpha expression. The Kaplan-Meier analysis showed a significant difference in the overall survival time related to the percentage of topo IIalpha (P<0.001) and Ki-67 (P<0.001) positive tumor cells. Multivariate Cox regression analysis revealed the expression of topo IIalpha as the most important prognostic factor (P<0.001) in MCL superior to the international prognostic index (IPI), the Ki-67 index and other clinical characteristics.

Down-regulation of human topoisomerase IIalpha correlates with altered expression of transcriptional regulators NF-YA and Sp1.

Topoisomerase IIalpha (Topo IIalpha) is an essential nuclear enzyme with a role in the maintenance of DNA topology. Topo IIalpha is a target for several anticancer drugs and the levels of activity of this enzyme have been implicated in the development of drug resistance. Our objective was to identify regulatory transcription factors involved in drug-induced down-regulation of Topo IIalpha. A breast cancer cell line was subjected to a pulsed exposure of doxorubicin and resistant clones propagated. Whole-cell extracts were studied by immunoblotting and RT-PCR for drug-induced changes in the amounts Topo IIalpha, Sp1, Sp3, NF-Y and MDR1. Topo IIalpha levels were reduced in six out of eight cell lines. Of these, three showed concomitant changes in the expression of Sp1 and NF-YA. Thus, we provide the first evidence for roles of Sp1 and NF-Y in bringing about the drug-induced down-regulation of Topo IIalpha gene expression.

Unique cell membrane expression of topoisomerase-II alpha as a useful diagnostic marker of liposarcoma.

Topoisomerase-II alpha (Topo-II alpha) is known as a cell cycle-related intranuclear marker. To the best of our knowledge, the expression of Topo-II alpha on extranuclear sites has not been reported. The aim of the present study was to determine the usefulness of Topo-II alpha immunostaining for detecting the lipoblasts that are essential to diagnosing liposarcoma. Surgical specimens, including benign lipomatous tumors (four cases), well-differentiated liposarcomas (three cases), myxoid liposarcomas (six cases), pleomorphic liposarcomas (two cases), dedifferentiated liposarcomas (two cases), myxoid malignant fibrous histiocytomas (six cases), and one case of mesenteric panniculitis, were studied. Samples were immunostained using antibodies for Topo-II alpha, S-100 protein and Ki-67. In addition, we used the western blot method to investigate immunohistochemical-affinity in adipocytes. Mature adipocytes and lipoblasts in all of the benign and malignant lipomatous tumors intensively expressed cell contours positivity for Topo-II alpha. Cytoplasm of the lipoblasts occasionally reacted to the antibody and highlighted intracytoplasmic small unilocular, multivacuolated, or bubble-like patterns. Western blot analysis confirmed a 70 kDa product reactive to Topo-II alpha in the cell membrane fragment of mature adipocytes. S-100 protein expressed adipocytes and lipoblasts, but the detection of lipoblasts was not as easy as in Topo-II alpha immunostaining. Immunoreactivity of Ki-67 was limited to the nuclei, and the nuclear labeling index of Ki-67 correlated with that of Topo-II alpha. The immunoreactivity of Topo-II alpha for lipoblasts was more sensitive and obvious than those of S-100 protein. Immunostaining using the antibody for Topo-II alpha seems to be useful in recognizing lipoblasts that have been overlooked in hematoxylin-eosin-stained preparations, and is a useful marker for diagnosing liposarcoma.

Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition.

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoII alpha gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21Cip1/WAF1 directly led to ICBP90 reduction in p53-/- HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

Anti‐DNA topoisomerase IIα autoantibodies in localized scleroderma

To determine the prevalence and clinical correlation of anti-DNA topoisomerase IIalpha (anti-topo IIalpha) antibody in patients with localized scleroderma. Anti-topo IIalpha antibodies or anti-DNA topoisomerase I (topo I) antibodies were determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Inhibition of topo IIalpha enzymatic activity by the antibodies was evaluated by decatenation assays using kinetoplast DNA as a substrate. IgG or IgM anti-topo IIalpha antibody was detected in 76% (35 of 46) of patients with localized scleroderma, and in 85% (11 of 13) of patients with generalized morphea, the severest form of localized scleroderma. This prevalence of the antibody in patients with localized scleroderma was much higher than that found in patients with systemic sclerosis (SSc) (5 of 37 [14%]), systemic lupus erythematosus (2 of 26 [8%]), dermatomyositis (2 of 20 [10%]), and in healthy controls (3 of 42 [7%]). Immunoblotting confirmed the presence of IgG anti-topo IIalpha antibody in sera from patients with localized scleroderma and showed no cross-reactivity of anti-topo IIalpha antibody with topo I. Anti-topo I antibody was not detected by ELISA in any sera from patients with localized scleroderma. In addition, anti-topo I antibody from SSc patients did not cross-react with topo IIalpha. The presence of anti-topo IIalpha antibody was associated with a greater total number of sclerotic lesions and number of plaque lesions in patients with localized scleroderma. Furthermore, anti-topo IIalpha antibody was able to inhibit topo IIalpha enzymatic activity. The results of the present study indicate that anti-topo IIalpha is a major autoantibody in localized scleroderma, and is distinct from anti-topo I antibody in SSc.

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

Differences in the longevity of topo IIalpha and topo IIbeta drug-stabilized cleavable complexes and the relationship to drug sensitivity.

DNA topoisomerase II (topo II) is an important cellular target for chemotherapeutic agents. Human cells have two isoforms of topo II (alpha and beta), and both are inhibited by the chemotherapeutic agents etoposide, amsacrine (mAMSA) and mitoxantrone. It is known that the cytotoxic importance of topo IIalpha or topo IIbeta drug-induced complexes differs depending on which drug is present. This study was designed to (a) assess isoform-specific formation and reversal of topo IIalpha and beta cleavable complexes, and (b) determine whether the cytotoxic importance of either isoform was related to differences in the longevity of the complexes. Mouse embryonic fibroblasts (MEFs) were used to study the cellular response to the topo II poisons etoposide, mitoxantrone and mAMSA. The longevity of topo IIalpha and beta complexes was determined using the TARDIS assay. This immunofluorescence assay can differentiate between the topo II isoforms and thus allowed us to investigate the persistence and importance of topo IIalpha and beta complexes for the first time. In MEFs treated with etoposide, 50% of topo IIalpha complexes dissociated within 40 min whereas dissociation of topo IIbeta complexes took only 20 min. Disappearance of complexes was a slower process for mitoxantrone-treated cells. The time taken to reduce topo IIalpha and topo IIbeta cleavable complexes by 50% was 10 and 6 h, respectively. In contrast, mAMSA-stabilized topo IIalpha and topo IIbeta cleavable complexes were equally stable (dissociation within 15 min for both isoforms). These stability data were confirmed using an in vitro assay. We previously demonstrated that topo IIalpha is the major target for etoposide and mitoxantrone but that both topo IIalpha and topo IIbeta are important for mAMSA cytotoxicity. The longevity of the topo IIalpha and beta cleavable complexes shown here is therefore an important factor in determining the cytotoxic sensitivity of either isoform to these drugs.

Evaluation of MDR1, LRP, MRP, and topoisomerase IIα gene mRNA transcripts before and after interferon-α, and correlation with the mRNA expression level of the telomerase subunits hTERT and TEP1 in five unselected human melanoma cell lines

Intrinsic and acquired multidrug-resistance (MDR) and the activity of the enzyme telomerase have been demonstrated in human melanoma. A direct regulation of the MDR pathways and of telomerase by interpheron-alpha (IFN-alpha), which is currently used in the therapy of advanced cutaneous melanoma, has also been hypothesized. In this study, we used five melanoma cell lines not selected in vitro for drug resistance (Me665/2/21, Me665/2/60, HT-144, SK-MEL-28, and SK-MEL-5), which in a previous study, had shown different responses to IFN-alpha in terms of proliferation, apoptosis, telomerase activity and expression of mRNA for the human telomerase reverse transcriptase (hTERT). We investigated the expression of the multidrug resistance (MDR1) gene, multidrug resistance protein (MRP), lung resistance protein (LRP), topoisomerase IIalpha (Topo IIalpha), hTERT, and telomerase-associated protein (TEP1), which is shared by telomerase and vault MDR proteins at the mRNA expression level, using the reverse transcription-PCR (RT-PCR). All cell lines showed an intrinsic expression of hTERT, TEP1, and MDR gene transcripts (only MDR1 mRNA was under the detection level in SK-MEL-28 cells). After IFN-alpha exposure, we observed either no effect, a trend towards a decrease of hTERT, MRP, and Topo IIalpha, or an increase of TEP1, MDR1, and LRP mRNA expression in some cell lines. Effects were usually temporary and not always significant. No correlation was found between hTERT and TEP1 mRNA expression, whereas significant positive correlations were found between TEP1 and MDR1 mRNA, and between TEP1 and LRP mRNA. IFN-alpha modulates differently MDR gene transcripts in human melanoma cell lines. Positive correlation between TEP1 and LRP also seems to identify them as common targets of IFN-alpha effects.

Enforced cytokinesis without complete nuclear division in embryonic cells depleting the activity of DNA topoisomerase IIalpha.

There are two distinct DNA topoisomerase II (topo II) isoforms, designated topo IIalpha and topo IIbeta, in mammalian cells. The function of topo IIalpha in the development of mammalian cells has not been elucidated because of a lack of topo IIalpha mutants. We generated mice with a targeted disruption of the topo IIalpha gene. The development of topo IIalpha-/- embryos was terminated at the 4- or 8-cell stage. When wild-type embryos at the 2- or 4-cell stage were treated with ICRF-193, a catalytic inhibitor of topo II, nuclear division occurred followed by cytokinesis to form 4 or 8 cells, respectively, then development was terminated. Microscope analysis of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei of both topo IIalpha-/- and ICFR-193-treated embryonic cells revealed a droplet-like structure connecting the terminals of two adjacent nuclei forming a bridge-like structure. Phosphorylated histone H3, a marker for the M phases, disappeared from the nuclei of the topo IIalpha-depleted embryonic cells. Laser scanning cytometry of the topo IIalpha-depleted cells revealed the presence of 2N DNA cells. Our results indicate that topo IIalpha has an essential role in the early stages of mouse development and that depletion of topo IIalpha from the embryonic cells causes incomplete nuclear division followed by enforced cytokinesis.

Nuclear factor-Y binding to the topoisomerase IIalpha promoter is inhibited by both the p53 tumor suppressor and anticancer drugs.

Expression of the human DNA topoisomerase IIalpha (topo IIalpha) gene is positively regulated by the binding of the nuclear factor Y (NF-Y) transcription factor to four of five inverted CCAAT boxes (ICBs) located in its promoter. We have demonstrated previously that expression of the p53 tumor suppressor inhibits human topo IIalpha promoter activity in murine (10)1 cells. In this report, we demonstrate that the inhibition of topo IIalpha gene expression by wild-type p53 correlates with the decreased binding of the transcription factor NF-Y to the first four ICBs of the topo IIalpha promoter. The expression of mutant p53 does not affect the binding of NF-Y. In NIH3T3 cells, we show that topo II-targeted drugs inhibit the binding of NF-Y to ICB sites in the topo IIalpha promoter. This effect is seen not only with drugs that result in DNA strand breaks but also with drugs that inhibit the catalytic activity of topo II, and even with the mitotic spindle inhibitor, vinblastine. Further experiments with p53-null (10)1 cells treated with these same drugs also demonstrate decreased NF-Y binding to the topo IIalpha ICBs. The data presented points to the existence of both p53-dependent and -independent mechanisms for regulating NF-Y binding to ICBs in the topo IIalpha promoter and thus the modulation of topo IIalpha gene expression.

Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate.

The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

Expression of topoisomerase IIalpha, Ki-67, proliferating cell nuclear antigen, p53, and argyrophilic nucleolar organizer regions in vulvar squamous lesions.

It was the aim of this study to investigate the expression of topoisomerase IIalpha (topo IIalpha), Ki-67, proliferating cell nuclear antigen (PCNA), p53, and argyrophilic nucleolar organizer region (AgNOR) staining in normal vulvar epithelia (NE, N = 10), vulvar condylomas (VC, N = 24), vulvar intraepithelial neoplasia (VIN, N = 26), as well as squamous cell carcinomas (SCC, N = 22) of the vulva. Formalin-fixed, paraffin-embedded archival tissue sections were immunostained with monoclonal antibodies against topo IIalpha, p53, and PCNA, as well as an affinity-isolated prediluted ready-to-use Ki-67 antibody using a standard immunohistochemical method, and stained with a colloid silver solution for AgNORs. Immunostaining was quantitated by determining the percentage of positively staining nuclei in each sample to express the labeling indices (LIs) by counting the immunoreactive nuclei in 1000 epithelial cells per case for each antibody. In each specimen 200 nuclei were examined using a x100 oil emersion lens, and the mean number of AgNORs per nucleus (AC) was calculated. The LIs for topo IIalpha, Ki-67, and PCNA as well as ACs increased stepwise from NE to VCs, VIN lesions, and SCCs. In contrast to PCNA LIs and ACs, a consistent correlation in all four groups was found for Ki-67 and topo IIalpha, suggesting that the latter is a proliferation-associated marker in these tissues. p53 expression was seen 8.3% of VCs, 30.8% of VIN lesions, and 54.45% of SCCs. p53 LIs were not correlated with LIs for topo IIalpha or Ki-67 in SCCs. The LIs for topo IIalpha, Ki-67, PCNA, p53, and ACs were not related to tumor progression, FIGO stage, or tumor grade in SCCs. This study presents topo IIalpha and Ki-67 as useful proliferation-associated markers of vulvar epithelia.