Differential expression of the topoisomerase II alpha and beta genes in human breast cancers.

Topoisomerase II is a key target for several anti-cancer drugs used for breast cancer therapy, including doxorubicin, epirubicin and mitoxantrone. Two isoforms of topoisomerase II (alpha and beta) have been described in human cells which differ in their subcellular localisation, biochemical properties and susceptibility to inhibition by anti-cancer drugs. The relative level of expression of the alpha and beta isoforms may contribute to the degree of tumour responsiveness to different chemotherapeutic agents. To assess the relationship between expression of topoisomerase II isoforms and established prognostic factors and pathological variables, 56 primary breast tumour samples were studied. The expression of the two topoisomerase II genes was apparently not co-ordinately regulated in these tissue samples. There was no relationship between any of the commonly used pathological variables [tumour size, lymph node status, S-phase fraction (SPF)] and the level of expression of topoisomerase II beta mRNA. However, high topoisomerase II alpha gene expression was significantly associated with a high SPF (sign-rank test; P = 0.01). Moreover, the ratio of mRNA levels for topoisomerase II alpha and beta showed a stronger relationship to SPF (median raito 0.62 for tumours with SPF < 10, and 1.64 for SPF > 10; P = 0.0021, sign-rank test). As expected from previous studies, an SPF > 10 was associated with poor overall survival (P = 0.01). Immunohistochemical analysis revealed that topoisomerase II beta was widely distributed ( > 90% positive tumour cells), but that topoisomerase II alpha expression was less widely expressed, with a pattern of expression similar to that of the proliferation-dependent antigen recognised by Ki67. Because topoisomerase II gene expression showed a log-normal distribution, log-transformed data were used in multivariate analysis of relapse-free survival. This showed that lymph node status and topoisomerase II beta mRNA expression were the only significant survival factors (P = 0.001 and 0.05, respectively, with relative risks of 1.3 and 1.8). These results indicate that topoisomerase II alpha, but not beta, expression is dependent upon cellular proliferation status, but that the more widely expressed topoisomerase II beta protein may play a significant role as a target for anti-tumour therapy.     

Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes.

Human topoisomerase II enzymes are targets for a number of widely used anticancer agents. We have analysed a lung adenocarcinoma cell line CALU3, which has co-amplified topoisomerase II alpha and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase II alpha and beta. The region of chromosome 17q amplified in CALU3 also includes the retinoic acid receptor alpha locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase II alpha and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase II alpha sequences to a cluster on one chromosome with single copies localised to others. CALU3 express high levels of topoisomerase II alpha is determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide. Topoisomerase II beta expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase II alpha in adenocarcinomas.     

Enhanced expression of DNA topoisomerase II by recombinant human granulocyte colony-stimulating factor in human leukemia cells.

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on DNA topoisomerase II (topo II) expression was studied in two human acute myelogenous leukemia cell lines, NKM-1 and NOMO-1, which express G-CSF receptor and proliferate in response to exogenous G-CSF. Northern blot analysis revealed that the level of topo II mRNA in 16-h stimulated cells in serum-free medium with G-CSF (10 ng/ml) was approximately 2-fold higher than that in cells without G-CSF. Enhanced topo II mRNA expression was detectable within 3 h after the addition of G-CSF. Topo II activity in crude nuclear extracts from 16-h G-CSF-stimulated cells was also found to be approximately 2-fold greater than that from unstimulated cells. According to in vitro cytotoxic assay, the sensitivity of G-CSF-stimulated cells to intercalating (daunorubicin) and nonintercalating (etoposide) topo II-targeting drugs increased significantly, whereas no enhancement of sensitivity was observed with an alkylating agent (4-hydroperoxycyclophosphamide). The augmented drug sensitivity observed was not due to the increased level of drug transport, as suggested by the similar extent of [3H]etoposide uptake between G-CSF-stimulated and unstimulated cells. By measuring the topo II mRNA and the cytotoxicity of the above mentioned drugs, we obtained essentially the same results in G-CSF-responsive leukemia cells isolated from three acute myeloblastic leukemia patients, as observed in the cultured cell lines. These findings strongly suggest that the sensitivity to "topo II-targeting drugs" could be augmented by exogenous G-CSF through elevated topo II activity in G-CSF-responsive leukemia cells.     

Correlation between DNA topoisomerase II alpha expression and sensitivity to etoposide in human glioma cell lines

The expression of topoisomerase II alpha (Topo II alpha) was investigated in six human glioma cell lines as a function of doubling time, cell cycle distribution, and sensitivity to an antineoplastic agent, etoposide (ETP). The Topo II alpha level was determined by immunohistochemical and flow cytometric studies using monoclonal antibody, 8D2, specific for Topo II alpha. In all cell lines, the Topo II alpha level correlated directly with sensitivity to ETP, but not with doubling time or percentage of cells in any phase of the cell cycle. These findings suggest that the cytocidal activity of ETP may be mediated by qualitative and quantitative changes in Topo II alpha in human glioma cells. We speculate that better knowledge of Topo II alpha expression in surgical specimens may lead to more individualized use of ETP in patients with malignant glioma.     

Investigation of human papillomavirus DNA in colorectal carcinomas and adenomas

Human papillomavirus (HPV) has been considered to be an etiological agent for anogenital cancers, such as cervical cancer and possibly a subset of cancers of the aerodigestive tract. The aim of the study was to evaluate the presence of human papillomavirus DNA in colorectal carcinomas and adenomas. Formalin-fixed and paraffin-embedded archival tissue samples were used for DNA extraction. One hundred and six colorectal carcinomas and 62 adenomas were screened by nested polymerase chain reaction (PCR) for HPV DNA with a control group of 49 cervical tissues with invasive cervical carcinoma and cervical intraepithelial neoplasia (CIN). In the study group, we did not find HPV DNA positivity in any of all the colorectal carcinomas and adenomas. In the control group with cervical lesions, 34 out of 49 (69.4%) samples were positive for the HPV DNA. These results indicated that there was no correlation between HPV infection and colorectal carcinomas and adenomas.     

Topoisomerase II alpha and topoisomerase II beta genes: characterization and mapping to human chromosomes 17 and 3, respectively.

Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.     

Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells

The intracellular level of DNA topoisomerase II appears to be reversibly regulated by serum concentration in cultured primary human skin fibroblasts (HSF). Upon serum starvation, the intracellular level of topoisomerase II in HSF, as monitored by immunoblotting with antitopoisomerase II antibodies, gradually decreased to a nondetectable level (less than 10(4) copies/cell) over a period of 72 h. Addition of 10% serum to the starved cells led to a gradual increase of the intracellular topoisomerase II to the original level (approximately 10(6) copies/cell) over a period of 24 h. The intracellular DNA topoisomerase II level in HSF is also sensitive to cell density; minimally a 7-fold decrease was observed when HSF were grown to saturation density in a constant serum concentration. Similarly, the intracellular levels of DNA topoisomerase II in other "nontransformed" cells such as mouse NIH 3T3 and 3T6 cells are also sensitive to both the serum concentration and the cell density. In contrast, topoisomerase II levels in transformed cells such as HeLa cells, L1210 cells, and SV40 T-antigen-transformed COS-1 cells are maintained at high levels (approximately 10(6) copies/cell) and are much less sensitive to growth conditions. The topoisomerase II level in HeLa cells synchronized by a double thymidine block remained relatively constant (less than 2-fold difference) throughout the late G1, S, G2, and M phases of the cell cycle. Our results suggest that the level of DNA topoisomerase II is primarily regulated in the G0-G1 phase of the cell cycle and is elevated to a high level (approximately 10(6) copies/cell) in proliferating cells. In contrast, the intracellular levels of DNA topoisomerase I in these cells were largely unaffected by these growth conditions either in HSF or in HeLa cells.     

DNA topoisomerase II immunostaining in human leukemia and rhabdomyosarcoma cell lines and their responses to topoisomerase II inhibitors.

DNA topoisomerase II is an enzyme that affects nuclear structure and function and is the target of a number of anticancer drugs in clinical use, including teniposide (VM-26). We have used our polyclonal antisera that recognize both the M(r) 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II in cytospin preparations of drug-sensitive (CEM) and VM-26-resistant (CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. We have also examined the nuclear distribution of topoisomerase II in monolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. In the absence of drug, we observed a focal "patchy" staining of nuclear topoisomerase II in all cell lines, that was especially notable in the lymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26 under conditions that increase the number of covalent topoisomerase II-DNA complexes increased both the intensity and the homogeneity of nuclear topoisomerase II staining in a subpopulation of cells; focal staining was less evident after treatment with drug. These responses were roughly proportional to the concentration of VM-26 used and required only brief (approximately 25-min) incubation with drug. We also found that treatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-anisidide similarly increased the intensity and homogeneity of nuclear topoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)methanesulfon-o-anisidide and 1-beta-D-arabinofuranosylcytosine, agents that do not inhibit topoisomerase II, did not produce this effect. Finally, the VM-26-mediated alteration in topoisomerase II staining intensity and distribution was attenuated in proportion to the degree of VM-26 resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNA-topoisomerase covalent ("cleavable") complexes in intact cells. Our findings indicate that anti-topoisomerase II drugs, such as VM-26, have profound effects on the ability to detect topoisomerase II in the nucleus and provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.     

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIα catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIα to decatenate kDNA, so that it could be considered a TOPO II poison.     

Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines

In a previous study, we established camptothecin (CPT)-resistant cell lines, A549/CPT and HT-29/CPT, from human lung cancer A549 and human colon cancer HT-29. A549/CPT was shown to express similar amounts of DNA topoisomerase I (topo I) as the parental line, and HT-29/CPT was shown to express lower amounts of topo I than its parental line. DNA topoisomerases I and II are known to be functionally related. In the present study, the possible alterations in topo II expression were examined in these human CPT-resistant lines. In A549/CPT and HT-29/CPT, the cellular contents of topo II and its mRNA were elevated over that seen in each parental line. Nuclear extracts from A549/CPT and HT-29/CPT showed higher topo II activity than those from the corresponding parental lines when the same amounts of nuclear protein were used. Topo II was partially purified from HT-29 and HT-29/CPT by hydroxylapatite column chromatography, and the enzyme activities were compared. HT-29/CPT showed higher topo II activity in the hydroxylapatite column-eluted fractions than HT-29. These results indicate the possible activation of topo II expression in the CPT-resistant cell lines.     

Expression of the MDR1 gene in human gastric and colorectal carcinomas

We measured expression of the MDR1 gene (also known as the PGY1 gene) in the human gastrointestinal tract. MDR1 messenger RNA (mRNA) levels were elevated in 13 of 15 colorectal carcinoma specimens and in six of 13 gastric carcinoma specimens. Well-differentiated colorectal carcinomas contained significantly higher concentrations of MDR1 mRNA than moderately differentiated colorectal carcinomas. Similarly, moderately differentiated gastric carcinomas contained higher concentrations of MDR1 mRNA than poorly differentiated gastric carcinomas. MDR1 gene expression in normal colorectal and gastric tissues adjacent to carcinomas was similar to that in the carcinomas. MDR1 gene expression in xenografts of colorectal and gastric carcinomas in nude mice was also investigated. Elevated expression of the MDR1 gene was seen in only four of 18 xenografts of colorectal carcinoma and was not seen in any xenografts of gastric carcinoma. P-glycoprotein was distributed over the luminal surface of the colorectal carcinoma. These results imply that the higher levels of MDR1 mRNA found in well-differentiated carcinomas derived from colorectal tissues are the results of increased expression of the MDR1 gene in the luminal surface cells. The level of expression of the MDR1 gene in colorectal and gastric carcinomas appears to correlate with the degree of differentiation and also appears to be affected by transplantation into nude mice.     

Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors

Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9-acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide.     

Culprit and victim -- DNA topoisomerase II

The phylogenetic antiquity of DNA topoisomerases indicates their vital function. Structure and maintenance of genomic DNA depend on the activity of these enzymes, and without them DNA replication and cell division are impossible. Topoisomerase II alpha has therefore become the main target of many antitumour therapy regimens, even though the exact mechanism of cell killing remains elusive. The success of this approach is limited by the development of spontaneous resistance, and drug-induced DNA damage can increase malignancy. Nevertheless, the combined use of topoisomerase-inhibiting drugs with different mechanisms of action promises to improve particular treatment designs. The degree of topoisomerase II expression in tumours may predict the clinical course and responsiveness to therapy.     

DNA topoisomerase II as a potential factor in drug resistance of human malignancies

The stabilization of the cleavable complex between DNA topoisomerase II and DNA by adriamycin (ADR), as well as by other topoisomerase II-targeted drugs, is an essential step in a process associated with drug cytotoxicity. Unlike many other cell types, ADR does not produce DNA cleavage in the lymphocytes of chronic lymphocytic leukemia (CLL). The CLL lymphocytes have been identified as quiescent cells with an extremely low level of topoisomerase II. The low level of this enzyme could constitute a basis for a new mechanism of drug resistance operating not only in CLL, but perhaps in any slow growing cancer with a large population of quiescent cells. Other factors contributing to drug resistance could include changes in enzyme regulation or processing of the cleavable complex, or the presence of a "mutant" enzyme which renders cancer cells unresponsive to topoisomerase II-targeted drugs. Suggested strategies in drug development, aimed at the topoisomerase II-related drug resistance, could include 1) the selection of topoisomerase I as an alternative target for cancer chemotherapy, 2) the development of ADR analogs which, unlike ADR, stabilize the topoisomerase II-DNA complex with high efficiency, and 3) the search for agents enhancing the SOS-like repair response, presumably triggered by DNA topoisomerase-targeted drugs.     

Expression of cyclooxygenase-2 and DNA topoisomerase II alpha in precancerous and cancerous lesions of the oral mucosa

The involvement of cyclooxygenase (COX)-2 in oral carcinogenesis and outcome of the patients is not fully understood. To determine whether COX-2 expression could serve as an indicator for them, we examined the expression of COX-2 and DNA topoisomerase (DNA-Topo) II alpha as an index of cell proliferating activity in precancerous and cancerous lesions of the oral mucosa. A 164 samples composed of 60 intraepithelial dysplasias (IEDs), 12 carcinomas in situ (CISs), 72 squamous cell carcinomas (SCCs) including 12 early invasive SCCs, 10 undifferentiated carcinomas (UCs), and 10 epithelial hyperplasias (EHPs) in the oral mucosa were examined immunohistochemically for COX-2 and DNA-Topo II alpha. Normal squamous epithelium as the control showed no COX-2 expression, whereas 41% of IEDs, 67% of CISs, 74% of SCCs, and 86% of UCs demonstrated increased COX-2 expression with elevated DNA-Topo II alpha labeling index (LI). High COX-2 expression was also observed in 61% of EHPs, but DNA-Topo II alpha LI was very low. Increased expression of COX-2 protein correlated with elevated DNA-Topo II alpha LI, indicating that COX-2 may contribute to malignant transformation and tumor growth. These two enzyme activities were increased as T, N, and M categories and stages proceeded. The patients with high expression of both COX-2 and DNA-Topo II alpha showed poor prognosis. Our results suggested that COX-2 expression become a possible indicator in oral carcinogenesis and may reflect the outcome of the patients.     

Study of DNA topoisomerase II in ataxia-telangiectasia cells

We have provided evidence recently for a defect in DNA topoisomerase II in ataxia--telangiectasia (A-T) lymphoblastoid cells. This study was initiated to investigate in greater detail the nature of this defect. Southern hybridization analysis was carried out on DNA from control and A-T Epstein--Barr virus-transformed lymphoblastoid cells. The pattern of digestion, using several restriction enzymes, was the same in both cell types. Expression of topoisomerase II mRNA occurred to the same extent and there was no difference in the size of mRNA between the cell types. Western blot analysis revealed that the same amount of a major band of topoisomerase II protein was present in A-T and control cells but there was evidence for a reduced amount of a lower-molecular-weight form in A-T only. Extraction and purification did not lead to alteration in size of the enzyme or in amount recovered.     

Ataxia telangiectasia mutated‐dependent regulation of topoisomerase II alpha expression and sensitivity to topoisomerase II inhibitor

Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM‐dependent manner after DNA damage. In vitro kinase assay and site‐directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia‐derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM‐dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low‐dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment‐associated secondary leukemia following exposure to TOP2 inhibitors.