Niclosamide induces mitochondria fragmentation and promotes both apoptotic and autophagic cell death

Mitochondrial dynamics not only involves mitochondrial morphology but also mitochondrial biogenesis, mitochondrial distribution, and cell death. To identify specific regulators to mitochondria dynamics, we screened a chemical library and identified niclosamide as a potent inducer of mitochondria fission. Niclosamide promoted mitochondrial fragmentation but this was blocked by down-regulation of Drp1. Niclosamide treatment resulted in the disruption of mitochondria membrane potential and reduction of ATP levels. Moreover, niclosamide led to apoptotic cell death by caspase-3 activation. Interestingly, niclosamide also increased autophagic activity. Inhibition of autophagy suppressed niclosamide-induced cell death. Therefore, our findings suggest that niclosamide induces mitochondria fragmentation and may contribute to apoptotic and autophagic cell death.     

Conformational diseases and ER stress-mediated cell death: apoptotic cell death and autophagic cell death

The expanded polyglutamine (polyQ) tracts observed in autosomal dominant neurodegenerative disorders have the tendency to form intracellular aggregates, thus enhancing apoptotic cell death and the formation of autophagic vesicles. PolyQ accumulation inhibits the ER-associated degradation system (ERAD) resulting in reduced retrotranslocation from the ER and increased accumulation of misfolded proteins in the lumen of ER. Autophagy is an early cellular defense mechanism associated with ER stress, but prolonged ER stress may induce autophagic cell death, with destruction of cellular components and apoptotic cell death. Endoplasmic reticulum (ER) stress may be the key signal for both of these events.     

Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death

Background

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.

Principal Findings

This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.

Conclusions

Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.     

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species(ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membranebound vacuoles characteristic of autophagy followed by autophagic cell death(referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.     

Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells

BackgroundDrug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis.MethodsFor cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry.ResultsPhosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation.ConclusionOXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR.General significanceWith two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.     

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible “attack me” signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.Key words: Tetrahymena, conjugation, nuclear apoptosis, monodansylcadaverine, macroautophagy, phagocytosis marker, glycoconjugates, phosphatidylserine     

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.     

Cell death in Tetrahymena thermophila: new observations on culture conditions.

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.Head and neck squamous cell carcinoma (HNSCC), which occurs in the oral cavity, oropharynx, larynx and hypopharynx, is the sixth most common malignancy worldwide.¹ It affects 600 000 new patients each year, which accounts for over 90% of head and neck cancers.^{2, 3} The current preferred therapy for HNSCC is combined surgery, radiotherapy, chemotherapy and biotherapy; however, the 5-year survival rate is still <50%, and the long-term survival rate has only marginally improved.^{4, 5, 6} As an important hallmark of head and neck cancer, apoptosis resistance restricts the efficacy of traditional therapies.⁷ Survivin (also called BIRC5) inhibits apoptosis-related proteins, regulates cell division, relates to stress response and promotes tumor-associated angiogenesis in HNSCC.⁸ Survivin is also associated with high-grade and advanced HNSCC, poor survival, high recurrence rate and chemotherapy and radiation resistance. Therefore, targeting survivin is promisingly beneficial for head and neck cancer therapies.Sepantronium bromide (YM155) is a small imidazolium-based compound (1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide) that selectively suppresses survivin expression⁹ and displays potent anticancer activities against various types of cancer.^{10, 11, 12} Previous researches have focused on that YM155 induced the apoptosis by downregulating survivin in cancer cells.^{10, 13, 14} Recent studies including ours have demonstrated YM155 also triggered autophagy in cancer cells.^{15, 16, 17} Macroautophagy or autophagy is considered to be another type of programmed cell death wherein proteins are degraded by autophagosomes and lysosomes.¹⁸ Autophagy also has an important role in tumorigenesis.¹⁹ Autophagy shares several regulatory systems and common pathways with apoptosis; thus, autophagy is closely linked with apoptosis. Beclin1 (ATG6), an autophagy-specific gene that is essential for autophagosome induction and elongation, interacts with several apoptosis-related genes, such as bcl-2, bcl-xl and survivin.²⁰ Therefore, YM155 may not only induce the apoptosis but also affect the autophagy in HNSCC.The present study investigated the antitumor effects of YM155 on HNSCC in vitro and in vivo through dual induction of apoptotic and autophagic cell death. Although it specifically suppressed the expression of survivin, we here proved YM155 also targeted the mTOR signaling pathway, which was the principal regulator of cancer cell survival and autophagy. Most importantly, in an inducible tissue-specific spontaneous HNSCC mouse model with 100% penetrance by the combined deletion of Tgfbr1 and Pten (Tgfbr1/Pten 2cKO) in the oral mucosa²¹ with ubiquitous activation of the Akt/mTOR/survivin pathway,²² YM155 exerted significant therapeutic effects by delaying tumor onset and suppressing tumor growth. This finding coincided with the xenograft results. Finally, the effects of YM155 when combined with traditional chemotherapeutic agent were also determined.     

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.     

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.     

Cytoplasm mediates both development and oxidation-induced apoptotic cell death in mouse zygotes

Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175 microM H(2)O(2) that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P<0.01) in zygotes reconstituted from untreated pronuclei and H(2)O(2)-stressed cytoplasts than those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to that of H(2)O(2)-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand, rates of cleavage and development to morula and blastocyst of the reconstituted zygotes, derived from stressed pronuclei and untreated cytoplasm, were significantly increased (P<0.05), compared to those of H(2)O(2)-treated, control zygotes, indicating that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative stress. It is suggested that cytoplasm, most likely mitochondria, plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes.     

Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells

Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5^K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTOR-dependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL.     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death

Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (FADD). Down-regulation of Atg5 expression in HeLa cells suppresses cell death and vacuole formation induced by IFN-gamma. Inversely, ectopic expression of Atg5 using adenoviral delivery induces autophagic cell death. Deletion mapping analysis indicates that procell death activity resides in the middle and C-terminal region of Atg5. Cells harboring the accumulated vacuoles triggered by IFN-gamma or Atg5 expression become dead, and vacuole formation precedes cell death. 3-Methyladenine or expression of Atg5(K130R) mutant blocks both cell death and vacuole formation triggered by IFN-gamma, whereas benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) inhibits only cell death but not vacuole formation. Atg5 interacts with FADD via death domain in vitro and in vivo, and the Atg5-mediated cell death, but not vacuole formation, is blocked in FADD-deficient cells. These results suggest that Atg5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

Rate of phenotypic assortment in Tetrahymena thermophila.

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted.     

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

Macronuclear DNA molecules of Tetrahymena thermophila.

The physical organization of the DNA in the macronuclei of Tetrahymena thermophila was investigated by using alternating-orthogonal-field gel electrophoresis. The genome consisted of a spectrum of molecules with lengths ranging from less than 100 to in excess of 1,500 kilobase pairs. There were about 270 different macronuclear DNA molecules, with an average size of about 800 kilobase pairs. Specific genes were mapped and were generally found on macronuclear DNA molecules of the same size in different strains of T. thermophila. This indicates that the molecular mechanisms giving rise to the macronuclear DNA molecules were precise. The fragmentation process that gave rise to macronuclear DNA molecules occurred between 11 and 19 h after the initiation of conjugation.