Micronucleus in cervical intraepithelial lesions and carcinoma.

AIMS AND OBJECTIVES: To score and compare micronucleus (MN) in the whole spectrum of cervical lesions including normal, inflammatory, abnormal squamous cell of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and invasive cancer (IC) and to evaluate the role of MN as a biomarker in different pre-neoplastic and neoplastic lesions. MATERIALS AND METHODS: A total of 224 slides, comprised of normal (40), inflammatory (40), ASC-US (30), LSIL (38), HSIL (30) and IC (46), were studied. All the cases of HSIL, IC and ASC-US had histopathology. The LSIL, normal and inflammatory smears were again reviewed by 2 experienced cytopathologists independently. Two observers separately and independently counted the number of micronucleated cells per 1,000 of epithelial cells in oil immersion magnification (×100 objective) which was expressed as MN score per 1,000 cells. RESULTS: The mean MN scores±SD in normal, inflammatory, ASC-US, LSIL, HSIL and IC cases of cervical lesions were 1.02±1.59, 0.4250±0.71208, 2.87±2.21, 4.7368±5.62179, 21.30±17.18 and 18.50±9.54, respectively. MN scores of IC and HSIL were significantly high compared to the normal (p<0.000), the inflammatory (p<0.000), the ASC-US (p<0.000) and to the LSIL (p<0.000) group (analysis of variance test). LSIL showed significant difference with the normal (p=0.043), the inflammatory (p=0.019), the HSIL (p<0.000) and the IC (p<0.000) group but not with the ASC-US (p=0.342) group. CONCLUSIONS: MN scoring on the epithelial cells of cervix could be used as a biomarker in cancer screening. This is an easy, simple, reliable, reproducible and objective test which can be performed on routinely stained smears.

Nuclear dimorphism: two peas in a pod.

The macro- and micronuclei of Tetrahymena reside in the same cytoplasm but are about as different as night and day. This extreme case of nuclear dimorphism can now be partially attributed to differences in the subunit compositions of their nuclear pore complexes.

Two distinct repeat sequences of Nup98 nucleoporins characterize dual nuclei in the binucleated ciliate tetrahymena.

Ciliated protozoa have two functionally distinct nuclei, a micronucleus (MIC) and a macronucleus (MAC) [1]. These two nuclei are distinct in size, transcriptional activity, and division cycle control, proceeding with cycles of DNA replication and nuclear division at different times within the same cell [2, 3]. The structural basis generating functionally distinct nuclei remains unknown. Here, we show that, in Tetrahymena thermophila, the nuclear pore complexes (NPCs) of MIC and MAC are composed of different sets of nucleoporins. Among the 13 nucleoporins identified, Nup98 homologs were of interest because two out of the four homologs were localized exclusively in the MAC and the other two were localized exclusively in the MIC. The two MAC-localizing Nup98s contain repeats of GLFG [4]. In contrast, the two MIC-localizing Nup98s lack the GLFG repeats and instead contain a novel repeat signature of NIFN. Ectopic expression of a chimeric MIC-localizing Nup98 homolog bearing GLFG repeats obstructed the nuclear accumulation of MIC-specific nuclear proteins, and expression of a chimeric MAC-localizing Nup98 homolog bearing NIFN repeats obstructed the nuclear accumulation of MAC-specific nuclear proteins. These results suggest that Nup98s act as a barrier to misdirected localization of nucleus-specific proteins. Our findings provide the first evidence that the NPC contributes to nucleus-selective transport in ciliates.

Enhanced level of micronuclei in peripheral blood lymphocytes of patients treated for restenosis with 32P endovascular brachytherapy.

BACKGROUND: Restenosis is the complete occlusion of the blood vessel leading to such complications as ischemia/angina, myocardial infarction, and death. It can be managed by endovascular brachytherapy with both gamma and beta sources. Endovascular brachytherapy is performed worldwide on several thousands of cases per year. The gamma-emitter 192Ir as well as the beta-emitters 32P and 90Sr are mainly used. The dose to the occluded endothelial wall is 20 Gy. Interestingly, no information with respect to the dose absorbed by the blood during the course of the treatment exists. The aim of the present investigation was to verify if the micronucleus test is suitable to detect the dose absorbed by lymphocytes in the course of endovascular brachytherapy with 32P. MATERIALS AND METHODS: Blood was drawn from 16 patients immediately before and 1 day after the treatment. Frequencies of micronuclei were assessed. In order to ensure that the micronuclei did not arise due to fluoroscopy or reperfusion, we analyzed lymphocytes of 16 control patients who underwent interventional cardiology with balloon angioplasty only. RESULTS AND CONCLUSIONS: Enhanced frequencies of micronuclei were observed in lymphocytes of some donors following brachytherapy. No correlation could be detected between the level of induced micronuclei and the absorbed dose. Also, no effect of fluoroscopy or reperfusion was seen. Thus, although brachytherapy of restenosis with 32P leads to weak enhancement of the micronucleus frequency in lymphocytes, the effect was not seen in all patients; the reason for this heterogeneous response remains to be elucidated.

An immunofluorescence technique for staining ciliated protozoans: highlighting cytoplasmic microtubular arrays and stages of micronuclear meiosis.

Ciliated protozoans represent useful organisms for studying the processes involved in the induction and progression of meiosis. In this short report we describe a technique that has allowed us to examine different meiosis phases during the sexual reproduction of Blepharisma japonicum. In order to visualize the phases of meiosis, sexually reproducing pairs were stained by an enhanced technique of anti-tubuline indirect immunofluorescence. Meiotic micronuclei, particularly those showing metaphase spindles, were clearly highlighted. The technique also heavily decorates the main microtubular cytoplasmic arrays in Blepharisma.

Block in anaphase chromosome separation caused by a telomerase template mutation.

Telomeres are essential for chromosome stability, but their functions at specific cell-cycle stages are unknown. Telomeres are now shown to have a role in chromosome separation during mitosis. In telomeric DNA mutants of Tetrahymena thermophila, created by expression of a telomerase RNA with an altered template sequence, division of the germline nucleus was severely delayed or blocked in anaphase. The mutant chromatids failed to separate completely at the midzone, becoming stretched to up to twice their normal length. These results suggest a physical block in mutant telomere separation.

Directed positioning of micronuclei in Paramecium tetraurelia with laser tweezers: absence of detectible damage after manipulation.

Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of "slow growth mutations" after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.

Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei.

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested that it accumulates in whichever nucleus is replicating. The inability of the micronucleus to accumulate Tetrahymena H1 or heterologous nuclear proteins, even at a period in the cell cycle when it is accumulating H4, suggests that it has a specialized transport system. These studies demonstrate that although the mechanism for localizing proteins to nuclei is highly conserved among eukaryotes, it can differ between two porecontaining nuclei lying in the same cytoplasm.

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei.

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.

Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field.

This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells. Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density. The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs. The course of further changes in cell organization depends on the polarity of fused cells. Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements. In contrast, cortical integration of heteropolar fused cells is limited. Cytoskeletal integration is particularly promoted if the cells are incubated in non-growing conditions. Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy.

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.