MAB4197 Sigma-AldrichAnti-Topoisomerase II Antibody, clone KiS1

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

Exit from mitosis is controlled by silencing of the spindle assembly checkpoint (SAC). It is important that preceding exit, all sister chromatid pairs are correctly bioriented, and that residual catenation is resolved, permitting complete sister chromatid separation in the ensuing anaphase. Here we determine that the metaphase response to catenation in mammalian cells operates through PKCε. The PKCε-controlled pathway regulates exit from the SAC only when mitotic cells are challenged by retained catenation and this delayed exit is characterized by BubR1-high and Mad2-low kinetochores. In addition, we show that this pathway is necessary to facilitate resolution of retained catenanes in mitosis. When delayed by catenation in mitosis, inhibition of PKCε results in premature entry into anaphase with PICH-positive strands and chromosome bridging. These findings demonstrate the importance of PKCε-mediated regulation in protection from loss of chromosome integrity in cells failing to resolve catenation in G2.

The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm1(-/-) mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Decreased lung tumorigenesis in epFoxm1(-/-) mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2alpha (TOPO-2alpha), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2alpha mRNA and protein levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2alpha promoter region, indicating that TOPO-2alpha is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO-2alpha expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy.

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors (CFs) disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naïve chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the "erase-and-rebuild" process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper (Gao T, et al., Cell Res 2007; 17: 135-150.) that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.

The relationship between meiotic recombination events and different patterns of pairing and synapsis has been analysed in prophase I spermatocytes of the grasshopper Stethophyma grossum, which exhibit very unusual meiotic characteristics, namely (1) the three shortest bivalents achieve full synapsis and do not show chiasma localisation; (2) the remaining eight bivalents show restricted synapsis and proximal chiasma localisation, and (3) the X chromosome remains unsynapsed. We have studied by means of immunofluorescence the localisation of the phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks; the SMC3 cohesin subunit, which is thought to have a close relationship to the development of the axial element (a synaptonemal complex component); and the recombinase RAD51. We observed a marked nuclear polarization of both the maturation of SMC3 cohesin axis and the ulterior appearance of gamma-H2AX and RAD51 foci, these being exclusively restricted to those chromosomal regions that first form cohesin axis stretches. This polarised distribution of recombination events is maintained throughout prophase I over those autosomal regions that are undergoing, or about to undergo, synapsis. We propose that the restricted distribution of recombination events along the chromosomal axes in the spermatocytes is responsible for the incomplete presynaptic homologous alignment and, hence, for the partial synaptonemal complex formation displayed by most bivalents.

The use of chemotherapy as a form of primary treatment for breast cancer is increasing and, as a result, more resection specimens contain tumours which have been exposed to cytotoxic drugs. We have studied the effects of chemotherapy on the tumour morphology and various biological features of breast carcinoma in a group of 35 patients. These were a group who responded to treatment in a clinical study of the use of primary chemotherapy designed to reduce tumour bulk prior to surgery. Characteristic morphological changes, temporally related to the administration of cytotoxic agents, are seen. The malignant cells become enlarged with vacuolated cytoplasm and vesicular nuclei containing prominent nuclei; occasionally the nuclei were angular and hyperchromatic. These features are interpreted as degenerative in nature. In 15 cases sufficient material was present in the pretreatment biopsies to compare the grade of the tumours before and after chemotherapy: changes were found in six tumours. Cytotoxic drugs do not induce a consistent pattern of change in the proliferation and apoptotic indices of individual tumours, but there is a tendency to reduce proliferative activity over all the tumours as a group. It was also found that chemotherapy is capable of modifying the expression of the oncoproteins c-erbB-2 and p53 in a minority of cases of breast cancer, usually resulting in an acquisition of immunoreactive oncoprotein. It is important to be aware of these effects when studying breast carcinomas removed after chemotherapy.

Gangliogliomas are tumors composed of intimately admixed neuronal and glial components and account for approximately 1% of all brain tumors. Here we report the histopathological findings in 61 gangliogliomas. Epilepsy was the most common presenting symptom. Most gangliogliomas were located in the temporal lobes (74%). Thirteen percent of the gangliogliomas were associated with glioneuronal hamartias. There was considerable variation in neuronal size and density, presence of binucleated neurons, calcifications, desmoplasia, lymphocytic infiltrate, pilocytic differentiation, Rosenthal fibers, location, or histological uniformity. Fifteen percent of the gangliogliomas contained areas of purely astrocytic differentiation. All tumors were examined immunohistochemically for an aberrant p53 tumor suppressor gene product and for the presence of nuclear antigens associated with cell proliferation (Ki-67, Ki-S1, proliferating cell nuclear antigen). In 45 of 61 cases (74%) labeling indices for Ki-67 were less than 1%. Nuclear labeling for Ki-67 was observed exclusively in the astrocytic component. Gangliogliomas with very large neurons had higher Ki-67 labeling indices and occurred in younger patients than gangliogliomas with small- or intermediate-sized neurons. None of the tumors had an aberrant expression of p53. The observations suggest that gangliogliomas may arise from glioneuronal hamartias through neoplastic transformation of the astrocytic component.

There is considerable interest in immunohistochemical markers of proliferation which are suitable for use on routinely fixed clinical material. The novel proliferation-associated antibody Ki-S1 shows promise in this respect. In this study we have: (i) defined the pattern of Ki-S1 labelling relative to the cell cycle phase; (ii) investigated the labelling pattern with Ki-S1 on a human breast cell line (ZR75) under varying proliferative conditions induced by serum deprivation and refeeding; (iii) examined in a flow cytometric study Ki-S1 staining in archival, clinical breast carcinoma samples. In exponentially growing cells Ki-S1 showed a marked cell cycle phase-specific variation in staining intensity which increased linearly through the S-phase, was high in G2 and reached its peak in mitosis. Ki-S1 staining intensity mirrored the changes in proliferative activity of ZR75 cells during serum deprivation and refeeding. In a small series of human breast carcinoma, Ki-S1 staining intensity correlated with S-phase fraction (SPF) derived from DNA profiles. The antigen labelled by Ki-S1 is extremely robust, resisting degradation by fixation and by an aggressive enzymic tissue disaggregation method. Ki-S1 warrants further investigation as a proliferation-related marker, particularly for routine clinical application.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.

cDNA segments for DNA topoisomerase II were amplified from rat brain RNA after reverse transcription by the polymerase chain reaction, using degenerate oligonucleotide primers deduced from the conserved regions of topoisomerase II of higher eukaryotes. The cDNA product from a successful amplification was homogeneous in length but heterogeneous in sequence. Restriction mapping of the cloned cDNA fragments revealed that they consisted of two distinct sequence groups. DNA sequencing of representative clones from each group, designated A and B, showed that they are highly homologous to cDNAs of human topoisomerase II isoforms, alpha and beta, respectively. Northern blot analysis indicated that the transcript level for rat topoisomerase II alpha was high in embryonic brain and in the cerebellum of 2-day newborns, followed by rapid decrease to a undetectable level at 4 weeks after birth. In contrast, rat topoisomerase II beta transcript was present throughout the embryonic and postnatal stages. In the developing cerebellum, cells expressing topoisomerase II alpha were confirmed exclusively to the outer mitotic zone of the external granular layer, whereas the transcript of topoisomerase II beta was detected over the entire cortical region. These results clearly indicate that the isoform alpha is expressed only in proliferating cells. The differential expression of topoisomerase II isozymes was also observed among developed tissues. Therefore, the isozymes are most likely to be involved in the following different physiological processes: topoisomerase II alpha in cell proliferation, and topoisomerase II beta in some processes unrelated to cell proliferation.