Effect of Diets and Low Temperature Storage on Adult Performance and Immature Development of Phyllonorycter ringoniella in Laboratory.

The Asiatic apple leaf miner, Phyllonorycter ringoniella (Matsumura), is an important insect pest to apples. We developed a rearing method for P. ringoniella in the laboratory. Overwintering pupae were collected from our apple orchard, and crabapple seedlings were selected as oviposition substrate and food source for the larval development. The mean developmental period was 25.9 ± 0.49 days from egg to adult and the survival rate was 0.387 under 25 ± 1 °C, 65 ± 5% RH and a photoperiod of 14:10 (L:D) h. Mean egg length and egg width were 0.336 ± 0.0043 and 0.259 ± 0.0046 mm, respectively. Mean body length and head width increased from 1.070 ± 0.0245 and 0.180 ± 0.0021 mm in first instar larvae to 5.027 ± 0.0718 and 0.321 ± 0.0021 mm in fifth instar larvae, respectively. The mean weight of the pupae was 0.946 ± 0.0132 mg. The wingspan of male adult (6.280 ± 0.0639 mm) was significantly larger than female (6.040 ± 0.0753 mm). The mean fecundity (56.9 ± 8.58 eggs per female) and longevity (8.2 ± 0.55 days) of females was highest when they were provided with 10% honey solution, followed by 10% sugar, water, and control. The females preferred to lay eggs (56.5 ± 3.84%) near the secondary leaf vein in a leaf. The larval mortality increased linearly (R2 = 0.94) with the number of larvae per leaf. The mortality of pupae increased from 5.6 ± 4.01 to 51.1 ± 9.88% as storage periods at 4 °C increased from 0 to 105 days. The rearing method and its biological characteristics presented in this study will contribute to further studies on this pest insect.

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells.

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes.

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.

p53 dependent centrosome clustering prevents multipolar mitosis in tetraploid cells.

BACKGROUND: p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. PRINCIPAL FINDINGS: Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. CONCLUSIONS: p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.

The fate of micronucleated cells post X-irradiation detected by live cell imaging.

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.

Effects of age on segregation of the X and Y chromosomes in cultured lymphocytes from Chinese men.

Chromosome malsegregation in binucleated lymphocytes is a useful endpoint to evaluate age effect on genetic stability. However, the investigations on chromosome malsegregation in binucleated lymphocytes from Chinese are scarce. In this study, peripheral blood lymphocytes were collected from 14 old (60-70 years) and 10 young (22-26 years) healthy Chinese men. To detect malsegregation of the sex chromosomes, multi-color fluorescence in situ hybridization (FISH) was performed on binucleated lymphocytes, cytokinesis-blocked by cytochalasin B at the first mitosis after phytohaemagglutinin stimulation. Compared with that in young men, a significant increase in frequencies of loss of chromosome X (9.2 +/- 3.2 per thousand vs. 1.1 +/- 0.9 per thousand, P < 0.001) and Y (2.5 +/- 1.9 per thousand vs. 0.2 +/- 0.3 per thousand, P < 0.001) was found in old men. Similarly, nondisjunction of chromosome X (16.5 +/- 3.4 per thousand vs. 3.5 +/- 1.1 per thousand, P < 0.001) and Y (7.2 +/- 2.6 per thousand vs. 2.4 +/- 1.3 per thousand, P < 0.001) occurred more frequently in old men than in young men. Regardless of donor's age, nondisjunction is more prevalent than loss for both chromosome X and Y. The frequencies of observed simultaneous malsegregation were relatively higher than the expected, suggesting an association between malsegregation. These results indicated that in Chinese men, malsegregation of the sex chromosomes increases with age in an associated fashion, and nondisjunction accounts for the majority of spontaneous chromosome malsegregation.

Abnormal synapses and recombination in an azoospermic male carrier of a reciprocal translocation t(1;21).

OBJECTIVE: To study the meiotic abnormalities during prophase I in an azoospermic man with t(1;21) reciprocal translocation. DESIGN: Analysis of synapses, recombination, and transcription inactivation in a testicular biopsy sample. SETTING: Research laboratory. PATIENT(S): One azoospermic patient with t(1;21) and five men with normal spermatogenesis. INTERVENTION(S): Immunostaining for SCP3, MLH1, and gamma-H2AX/BRCA1 was performed on biopsy to identify synapses, recombination, and transcriptional inactivation, respectively. MAIN OUTCOME MEASURE(S): Synapses, recombination, and transcriptional inactivation in meiosis I. RESULT(S): The t(1;21) carrier had a larger number of synaptonemal complexes with gaps and a lower rate (46%) of XY pairs with MLH1 foci than the controls (78%). The asynapsed quadrivalents, which were often associated with an XY body (84%), were frequently observed (96%) in pachytene cells of the translocation carrier. The variant histone gamma-H2AX and BRCA1 proteins were found to be located at the asynapsed quadrivalents. CONCLUSION(S): These results suggest that impaired synaptic integrity of translocated chromosomes may affect synapses, recombination frequency of XY pairs, and transcriptional activation of asynapsed areas, and consequently may impair fertility in men.

Multiple origins of spontaneously arising micronuclei in HeLa cells: direct evidence from long-term live cell imaging.

Although micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their origins has not been completely addressed due to limitations of traditional methods. Recently, long-term live cell imaging was developed to monitor the dynamics of single cell in a real-time and high-throughput manner. In the present study, this state-of-the-art technique was employed to examine spontaneous micronucleus (MN) formation in untreated HeLa cells. We demonstrate that spontaneous MNi are derived from incorrectly aligned chromosomes in metaphase (displaced chromosomes, DCs), lagging chromosomes (LCs) and broken chromosome bridges (CBs) in later mitotic stages, but not nuclear buds in S phase. However, most of bipolar mitoses with DCs (91.29%), LCs (73.11%) and broken CBs (88.93%) did not give rise to MNi. Our data also show directly, for the first time, that MNi could originate spontaneously from (1) MNi already presented in the mother cells; (2) nuclear fragments that appeared during mitosis with CB; and (3) chromosomes being extruded into a minicell which fused with one of the daughter cells later. Quantitatively, most of MNi originated from LCs (63.66%), DCs (10.97%) and broken CBs (9.25%). Taken together, these direct evidences show that there are multiple origins for spontaneously arising MNi in HeLa cells and each mechanism contributes to overall MN formation to different extents.