Identification of tumor-associated antigens of lung cancer: SEREX combined with bioinformatics analysis.

The aim of this study is to identify novel tumor-associated antigens (TAAs) of lung cancer by using serological analysis of recombinant cDNA expression library (SEREX) and bioinformatics analysis as well as to explore their humoral immune response. SEREX and pathway enrichment analysis were used to immunoscreen TAAs of lung cancer and elaborate their function in biological pathways, respectively. Subsequently, the sera level of autoantibodies against the selected TAAs (TOP2A, TRIM37, HSP90AB1, EEF1G and TPP1) was detected by immunoserological analysis to explore the immune response of these antigens. The Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) database were applied to explore the mRNA and protein expression level of TOP2A, TRIM37 and HSP90AB1 in tissues, respectively. Seventy positive clones were identified by SEREX which contain 63 different genes, and 35 genes of them have been reported. These 35 genes were mainly related to regulation of different transcription factor and performed enrichment in legionellosis, RNA transport, IL-17 signaling pathway via enrichment analysis. Additionally, the positive rate of autoantibodies against TOP2A, TRIM37 and HSP90AB1 in lung cancer patients were typically higher than normal control (NC; P < 0.05). Moreover, the combination of the autoantibodies against TOP2A, TRIM37 and HSP90AB1 possessed an excellent diagnostic performance with sensitivity of 84% and specificity of 60%. The mRNA expression level of TOP2A was obviously unregulated in squamous cell carcinoma (SCC) tissues and adenocarcinoma (ADC) tissues compared to normal tissues (P < 0.05). In addition, TRIM37 and HSP90AB1 also showed a significant difference between SCC and NC at the mRNA expression level (P < 0.05). This study combining comprehensive autoantibody and gene expression assays has added to the growing list of lung cancer antigens, which may aid the development of diagnostic and immunotherapeutic targets for lung cancer patients.

Study of DNA topoisomerase IIa expression in canine lymphomas and its potential role as a marker of sensitivity to anthracycline-based chemotherapy in dogs.

INTRODUCTION: Canine lymphoma remains one of the most chemotherapy-responsive neoplasia in dogs. Many factors affect the prognosis in dogs treated for lymphoma, but indications for a specific treatment regimen in individual animals with lymphoma are poorly defined. Topoisomerase IIα (TOPIIα) is a key enzyme in DNA replication and a molecular target for TOPIIα inhibitors, including anthracyclines. The aim of this study was to determine the expression of TOPIIα in canine malignant lymphomas. The relationship between TOPIIα expression in canine lymphomas and potential sensitivity of neoplastic cells to anthracycline-based chemotherapy is discussed. MATERIALS AND METHOD: Samples of formalin-fixed paraffin-embedded lymph nodes from 47 dogs with different subtypes of non-Hodgkin's (34 B-cell and 13 T-cell) lymphoma were immunohistochemically labeled with anti-TOPIIα. The number of positive cells and the intensity of the reaction were taken into account in order to assess TOPIIα expression. RESULTS: TOPIIα expression was evident in all cases, although differences in the number of positive cells and intensity of the reaction were demonstrated between B-cell and T-cell lymphoma groups as well as within individual groups. Based on the established scoring system, in the B-cell lymphoma group statistically higher expression of TOPIIα was found compared to the T-cell lymphoma group (P = 0.006). In B-cell lymphoma group moderate (41.18%) and strong (32.35%) TOPIIα expression predominated, whereas among T-cell lymphoma group the majority were cases with a weak (46.15%) TOPIIα expression. CONCLUSION: These preliminary results indicate that further studies are needed to determine the prognostic value of TOPIIα expression with regard to the sensitivity of canine B-cell lymphomas to anthracycline-based chemotherapy regimen. Nevertheless, this study indicates the possibility of choosing the appropriate treatment of canine lymphoma based on TOPIIa expression.

Discovering novel lung cancer associated antigens and the utilization of their autoantibodies in detection of lung cancer.

OBJECTIVE: The identification of tumor-associated antigens (TAAs) and their corresponding autoantibodies in lung cancer (LC) may expand our vision of cancer immunity. This study aims to screen novel TAAs to distinguish LC from the healthy population. METHODS: In our previous study, 35 genes encoding LC-associated TAAs were identified from the serological analysis of recombinant cDNA expression libraries (SEREX), and Oncomine database was further used to identify potential genes in cancer progression. Autoantibody to TAAs were tested by enzyme-linked immunosorbent assay (ELISA) in sera from 1379 participants in validation set and verification set. FINDINGS: Based on analysis of three independent microarrays in Oncomine, ten genes were consistently dysregulated in LC. The sera level and positive frequency of the anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 from LC patients were higher than normal control in validation set. The area under curve (AUC) of anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 was respectively 0.758, 0.787, 0.707, 0.668. The sensitivity of these four autoantibodies for LC detection ranged from 26.63 % to 32.07 % with the specificity over 90 %. Data from the verification set confirmed the results. Except that, the frequency of serum autoantibody against TOP2A (43.3 %) and ACTR3 (50.0 %) was significantly higher in early stage LC than late stage (23.6 % and 22.3 %, respectively). CONCLUSION: TOP2A, ACTR3, RPS6KA5 and PSIP1 can elicit humoral immune response in LC and their autoantibodies have relationship with the tumorigenesis of LC. Anti-TOP2A and anti-ACTR3 have the potential to serve as a serological biomarkers in early stage LC.

Mutations in topoisomerase IIß result in a B cell immunodeficiency.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

ProEx C as Diagnostic Marker for Detection of Urothelial Carcinoma in Urinary Samples: A Review.

The gold standard for the detection of urothelial carcinoma is represented by urethro-cystoscopy and biopsy. Both procedures are invasive and expensive and therefore cytology is often used as first approach to investigate on a possible neoplasia, being a safe and cost-effective diagnostic modality of evaluation. Because cytology alone is not highly sensitive for detection of low grade urothelial carcinoma and recurrence of the disease, several adjunct markers and urine based tests for urothelial carcinoma have been developed, which can help in the final diagnosis. In particular, ProEx C is an immunohistochemical cocktail containing antibodies direct against topoisomerase IIα (TOP2A) and minichromosome maintenance 2 (MCM2) proteins. It proved to be a valid biomarker especially in detecting squamous intraepithelial lesions in cervical liquid-based samples and in discerning these lesions from their mimickers, as well as in ovarian, endometrial, vulvar, primary and metastatic melanomas, breast, pancreatic and renal cell carcinomas. This brief review covers the effective utility of ProEx C as adjunct tool in assessing the urothelial lesions in urine cytology, also providing prognostic and therapeutic information to help in clinical decisions.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 µg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.

Expression of ProEx C in primary and metastatic urothelial carcinoma.

BACKGROUND: ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. Both these proteins are over-expressed in the cell nucleus during aberrant S-phase induction of neoplastic cells, which leads to cell proliferation. The aim of this study was to determine whether ProEx C expression can detect primary and metastatic urothelial carcinoma (UC). METHODS: Thirty one fine needle aspiration cell blocks (CB) with metastatic UC were identified. Immunohistochemical staining for ProEx C and thrombomodulin was performed. Additionally, staining for Pro Ex C was also performed in tissue microarrays (TMA) of 46 cases of primary UC and carcinomas from colon (80), stomach (31), pancreas (33), liver (92), ovary (24), endometrium (25), breast (60), lung (27), kidney (32), and prostate (44), as well as melanoma (22). Nuclear staining of ProEx C and membrane staining of thrombomodulin in at least 10% tumor cells was considered a positive result. RESULTS: Both ProEx C and thrombomodulin have similar sensitivity for metastatic UC (84% vs. 77%, p=0.75; whereas ProEx C yielded a higher sensitivity of 93% for primary UC than thrombomodulin (72%, p=0.01). In addition to UC, ProEx C is also expressed in most of the malignant neoplasms tested in our TMA study, and has the highest sensitivity in colon and stomach carcinomas (94%). CONCLUSION: ProEx C has high sensitivity for UC. However, it is also expressed in carcinomas of colon, stomach, breast, and lung carcinomas and may not be a useful marker for workup of metastatic UC.

Functional cloning of recurrence-specific antigens identifies molecular targets to treat tumor relapse.

Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expressed cDNA library derived from recurrent tumor cells. By screening the protective, recurrence-derived VSV-cDNA library, here we identify topoisomerase-IIα (TOPO-IIα) as a recurrence-specific tumor antigen against which tolerance can be broken. Tumor recurrences, in two different types of tumor (prostate and melanoma), which had evaded two different frontline treatments (immunotherapy or chemotherapy), significantly overexpressed TOPO-IIα compared with their primary tumor counterparts, which conferred a novel sensitivity to doxorubicin (DOX) chemotherapy upon the recurrent tumors. This was exploited in vivo using combination therapies to cure mice, which would otherwise have relapsed, after suboptimal primary therapy in both models. Our data show that recurrent tumors-across histologies and primary treatments-express distinct antigens compared with the primary tumor which can be identified using the VSV-cDNA library technology. These results suggest that it may be possible to design a few common second-line therapies against a variety of tumor recurrences, in some cases using agents with no obvious activity against the primary tumor.

ProEx C as an adjunct marker to improve cytological detection of urothelial carcinoma in urinary specimens.

BACKGROUND: ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. ProEx C staining is being used to assist in diagnoses of the gynecological specimens. This study was designed to determine the utility of ProEx C in urine cytology samples for improving the detection of urothelial carcinomas where a significant number of urine cytology specimens are diagnosed as "atypia." METHODS: Sixty urinary specimens (12 negative, 13 positive, and 35 atypical cases) were stained with ProEx C, and ProEx C results were recorded as positive when nuclear staining was only seen in at least one morphologically atypical urothelial cell. RESULTS: All 12 benign cytology samples showed negative staining with ProEx C. Twelve of 13 cases that had a malignant cytologic diagnosis showed a positive nuclear staining of the malignant cells. Eighteen of 35 cases with atypical cytologic diagnoses showed positive nuclear staining. Of the 35 cases with "atypia," 17 had a malignant histopathologic follow-up. In this study, ProEx C stain had an overall sensitivity of 78.4%, specificity of 95.7%%, positive predictive value of 96.7%, and negative predictive value of 73.3% for the detection of urothelial carcinoma. CONCLUSIONS: ProEx C stain is a useful adjunct test to urine cytologic analysis, even in specimens with limited cellularity. In urinary smears, this test is most useful in stratification of the "atypical" diagnoses into benign and malignant subsets. To the authors' knowledge, this is the first study of ProEx C application in urine cytology as an adjunct marker for detection of urothelial carcinoma.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples.

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.

Comparison of p16INK4a and ProEx C immunostaining on cervical ThinPrep cytology and biopsy specimens.

ProEx C and p16(INK4a) staining of cytology/histology specimens have recently been explored to help distinguish high-grade squamous intraepithelial lesions (HSIL) from benign mimics. The goal of this study was to evaluate the performance characteristics of p16 and ProEx C in tissue and patient matched ThinPrep liquid-based cytology specimens. Residual cervical ThinPrep cytology specimens and tissue blocks (N = 64) from 63 patients were stained with p16 and ProEx C. Review of immunostained material, Papanicolaou and H&E stained slides was performed by two cytopathologists. The cytology slides were evaluated for the presence or absence of squamous atypia as well as immunoreactivity. Histologic specimens were interpreted as negative, indeterminate, or positive for each immunostain. There was 86% agreement (55/64) between the p16 and ProEx C stains on tissue specimens. Eleven specimens were interpreted as positive for both stains. All had a low- or high-grade squamous lesion on the corresponding H&E section. ProEx C was able to identify four low-grade squamous intraepithelial lesion specimens that were interpreted as negative by p16. All four HSIL specimens demonstrated p16 and ProEx C staining. However, 84% of cytology negative specimens demonstrated false-positive staining. Clinical utilization of both stains, combined with morphologic features, may be beneficial for confirming HSIL on histologic specimens. ProEx C and/or p16 immunostains may lead to a false-positive result in cytology specimens due to staining of normal appearing cells.

Topoisomerase II alpha as a universal tumor antigen: antitumor immunity in murine tumor models and H-2K(b)-restricted T cell epitope.

Topoisomerase II alpha (Top2alpha) is an attractive candidate to be used as a tumor antigen for cancer immunotherapy, because it is abundantly expressed in various tumors and serves as a target for a number of chemotherapeutic agents. In this study, we demonstrated the immunogenicity of Top2alpha, using dendritic cells (DC) electroporated with RNA encoding the Top2alpha C-terminus (Top2alphaCRNA/DC). Top2alphaCRNA/DC were able to demonstrate in vitro stimulation of T cells from mice that were previously vaccinated with Top2alpha-expressing tumor lysate-pulsed DC. Vaccination with Top2alphaCRNA/DC induced Top2alpha-specific T cell responses in vivo as well as antitumor effects in various murine tumor models including MC-38, B16F10, and GL26. DC pulsed with p1327 (DSDEDFSGL), defined as an epitope presented by H-2K(b), also induced Top2alpha-specific immune responses and antitumor effects. Based on these data, Top2alpha is suggested to be a universal target for cancer immunotherapy.

Is the expression pattern of BD ProExC the same as Ki-67? A comparative analysis in cervical biopsies.

BD ProExC (ProExC) and Ki-67, both used as biomarkers for high-grade cervical intraepithelial neoplasia (HG CIN), yield discordant staining in some cervical biopsies. This study compared ProExC and Ki-67 expression in 197 cervical biopsies with consensus diagnoses (55 negative, 21 atypical squamous metaplasia, 61 low-grade CIN, and 60 HG CIN). Percentages of immunostained nuclei were evaluated by 2 pathologists yielding 68 (35%) cases with discordant ProExC/Ki-67 immunostaining for analysis. In 78% of cases the difference in staining involved <25% of lesional cells. This was noted across all morphologic diagnoses, being most frequent in HG CIN. Discordant staining involving >50% of lesional cells occurred in 22% of discrepant cases, being most frequent in atypical squamous metaplasia. Using staining in >50% of lesional nuclei as a positive result, positive/negative discordance occurred in 25 cases (13% of all cases) including 18% of HG CIN cases. Fourteen cases were ProExC+ (7 of which were strongly p16+) and 11 were Ki-67+ (6 of which were strongly p16+).

Mitotic chromosome structure: reproducibility of folding and symmetry between sister chromatids.

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an approximately 1 microm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to approximately 300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.

Longitudinal clinical evaluation of a novel antibody cocktail for detection of high-grade squamous intraepithelial lesions on cervical cytology specimens.

CONTEXT: Although cervical cancer screening by cytology is successful, the test continues to show relatively poor operating characteristics. Cell cycle markers may enhance detection of high-grade squamous intraepithelial lesions. OBJECTIVE: To determine the clinical usefulness of ProExC, an immunocytochemical assay for cell cycle components, performed on routine cervical cytology samples. DESIGN: Cervical cytology samples were collected using the SurePath method. Residual cells remaining after preparation of the Papanicolaou-stained slide were used to make a second slide for ProExC staining using an indirect polymer-based immunoperoxidase method. Only adequately cellular slides were evaluated for the presence of nuclear staining within cytologically abnormal epithelial cells. Results were correlated with clinical follow-up. RESULTS: Six hundred twenty-four samples were satisfactorily cellular and stained. Correlation with clinical follow-up for subsequent cervical intraepithelial neoplasia 2+ on biopsy/high-grade squamous intraepithelial lesion on cytology (CIN 2+/HSIL) showed that 434 results were true negative, 78 true positive, 18 false-negative, and 94 false-positive, resulting in a sensitivity/specificity of 81%/82%. When ProExC results were combined with any level of cytologic atypia, sensitivity for CIN 2+/HSIL was 92% and specificity was 84%. CONCLUSIONS: ProExC shows promise as an aid in enhancing the sensitivity and specificity of cervical cytology for subsequent CIN 2+/HSIL and may be useful in identifying those cervical lesions most apt to progress.

DNA topoisomerases I and II in human mature sperm cells: characterization and unique properties.

BACKGROUND: The condensed state of the human sperm's chromatin is essential for the compact structure of the spermatozoon head, which is important for the fertilization process. The enzymes DNA topoisomerases (topo I and topo II) are responsible for the topological structure of the chromatin in somatic cells. The activities and the characterization of topoisomerases in mature human sperm cells have not been previously investigated. METHODS: Sperm cells were purified from the semen of healthy donors by standard procedures and assays measuring the activities, protein size and sensitivity to inhibitors of topoisomerases were performed. RESULTS: Topo I and topo II DNA relaxation activities are present in nuclear extracts derived from human sperm. The sperm topo I activity is inhibited by camptothecin, similarly to the somatic enzyme. An 85 kDa sperm protein, compared with the 100 kDa somatic topo IB enzyme, reacted with anti-human topo I antibody. Sperm topo II lacks the DNA decatenation activity of the somatic enzyme and a 97 kDa protein, compared with the 170 kDa somatic topo IIalpha enzyme, was detected with anti-human topo II antibody. Sperm nuclear extracts contained inhibitors of somatic topo II decatenation activity. CONCLUSIONS: Topoisomerase I and II activities as well as topo I and topo II proteins are present in mature human sperm cells. These enzymes possess unique properties compared with their somatic counterparts.