Autophagic Adaptations to Long-term Habitual Exercise in Cardiac Muscle.

Autophagy has been shown to be responsive to physical exercise. However, the effects of prolonged habitual exercise on autophagy in cardiac muscle remain unknown. The present study aimed to examine whether long-term habitual exercise alters the basal autophagic signalling in cardiac muscle. Female Sprague-Dawley rats aged 2 months were randomly assigned to control and exercise groups. Animals in exercise group were kept in cages with free access exercise wheels to perform habitual exercise for 5 months. Animals in the control group were placed in cages without exercise wheels. Ventricular muscle tissues were harvested for analysis after 5 months. Phosphorylation statuses of upstream autophagic regulatory proteins and protein expressions of downstream autophagic facts remained unchanged in the cardiac muscle of exercise animals when compared to control animals. Intriguingly, the protein abundance of microtubule-associated protein-1 light chain -3 II (LC3-II), heat shock protein 72 (HSP72) and peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α) were significantly increased in cardiac muscle of exercise rats relative to control rats. 5 months of habitual exercise causes the adaptive increase in LC3-II reserve without altering autophagic flux, which probably contributes to the elevation of cellular autophagic capacity and efficiency of cardiac muscle.

Effects of luzopeptins on protein B23 translocation and ribosomal RNA synthesis in HeLa cells.

Localization of protein B23 in HeLa cells after treatment with luzopeptin A and its analogues was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with luzopeptin A (50 ng/ml), luzopeptin B (500 ng/ml), or luzopeptin D (10 ng/ml) for 2 h, uniform nucleoplasmic rather than specific nucleolar fluorescence was observed. Luzopeptin C had no effect on protein B23 translocation. Luzopeptin D, A, and B inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with 50% inhibitory concentration values of 3.7 +/- 1.1 (SD), 10.8 +/- 2.1, and 122.0 +/- 34.0 ng/ml, respectively. Less than 10% inhibition of [3H]uridine incorporation was found with luzopeptin C (500 ng/ml and 2 h incubation). Ribosomal RNAs (28 and 18S) were isolated from HeLa cells treated with luzopeptin D (50 ng/ml; 2 h). They were then separated and analyzed in 1% agarose gel electrophoresis. There were 90.1 +/- 1.38 and 95.0 +/- 1.04% inhibition of [3H]uridine incorporation into 28 and 18S ribosomal RNA, respectively. The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was luzopeptin D greater than luzopeptin A greater than luzopeptin B much greater than luzopeptin C, which correlates with the order of their 50% inhibitory concentration values for inhibition of [3H]uridine incorporation. With 34-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. With 70-85% inhibition of RNA synthesis, a uniform nucleoplasmic fluorescence was observed. These results indicate that translocation of protein B23 as observed by indirect immunofluorescence may be a rapid and simple screening test for the selection of antitumor agents which inhibit ribosomal RNA synthesis.

Nucleolar protein B23 translocation after doxorubicin treatment in murine tumor cells.

Rats bearing Novikoff hepatoma ascites cells were given i.p. injections of actinomycin D, doxorubicin, or daunorubicin. Four hours after injection, tumor cells were removed from the ascites fluid and analyzed for protein B23 translocation using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. Treatment with actinomycin D (1.25 mg/kg), doxorubicin (25 mg/kg), or daunorubicin (12.5 mg/kg) produced a uniform nucleoplasmic fluorescence. This change in immunofluorescence distribution indicated that protein B23 translocated from the nucleolus to the nucleoplasm after drug treatment. These results are an extension of previous studies with HeLa cells (Yung et al., Cancer Res. 46: 922-925, 1986). Doxorubicin-resistant and -sensitive mouse leukemia cells (P388) were cultured in medium containing various doses of doxorubicin for 4 h, and the responsive levels of the cells to doxorubicin were compared. At 50 micrograms/ml doxorubicin, 86% of the doxorubicin-sensitive cells showed uniform nucleoplasmic fluorescence, and less than 2% of the cells retained nucleolar fluorescence. At this same dose, only 9% of the resistant cells showed nucleoplasmic fluorescence, and 75% of the cells retained nucleolar fluorescence. At 100 micrograms/ml, about 26% of the resistant cells showed translocation, in contrast to 100% of the sensitive cells that showed B23 translocation. About 57% of the resistant cells showed an intermediate effect, and about 17% of the resistant cells maintained bright nucleolar fluorescence at this dose. The resistant cells also showed less responsiveness to actinomycin D. These results suggest that identification of "B23 translocation" may be used to detect drug-resistant cells and to study the efficacy of certain antitumor agents.

Interaction of antileukemia agents adriamycin and daunomycin with sphinganine on the differentiation of human leukemia cell line HL-60.

A slight induction of granulocytic differentiation of HL-60 cells occurred after treatment with antileukemia chemotherapeutic agents Adriamycin (ADM) and daunomycin (DM). Addition of an inhibitor (sphinganine, SP) of protein kinase C (PKC) enhanced 2-4-fold the ADM or DM-induced differentiation. This phenomenon was accompanied by a slightly augmented antiproliferative effect. The enhancement of differentiation induction in these treatments seemed to be absolute, since the combination treatment (ADM-SP or DM-SP) showed about 2.5-3.6 times as many differentiated cells as the treatment with the anticancer drugs ADM or DM alone. Further characterization of the interaction of ADM and DM with SP on differentiation of HL-60 cells was carried out. Whereas the addition of SP in the fresh medium after the removal of ADM or DM (0.5 h treatment) enhanced the induction of differentiation, a pretreatment (24 h) of the cells with SP followed by continuous exposure to ADM or DM did not show such enhancement effect. The addition of SP at as late as 48 h after the administration of ADM or DM potentiated the induction of differentiation to the same extent as in the simultaneous combination of ADM-SP or DM-SP. Similar results were obtained in the experiments with another PKC inhibitor, staurosporine. These results indicated that inhibition of PKC activities may play an important role in the later events during the induction of differentiation elicited by ADM or DM. The use of the antileukemia drugs ADM and DM in combination with an inhibition of PKC activity results in enhancement of induction of differentiation and suggests a new strategy and a promising approach to the treatment of leukemia.

Short exposure to actinomycin D induces "reversible" translocation of protein B23 as well as "reversible" inhibition of cell growth and RNA synthesis in HeLa cells.

HeLa cells were grown in medium containing various amounts of actinomycin D for various times. Cellular localization of protein B23 was detected using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. A shifting of nucleolar to nuclear fluorescence was observed with increasing doses of actinomycin D and longer incubation times. The degree of translocation of protein B23 from nucleoli to nucleoplasm is dependent on the amount of the drug used and the duration of incubation. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.01-0.25 microgram/ml) induced "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. A majority of cells (greater than 75%) treated with actinomycin D (0.01-0.25 microgram/ml) for 0.5 h still retained bright nucleolar fluorescence. A shifting of nucleolar to nuclear fluorescence as well as inhibition of cell growth and RNA synthesis were observed within 6 h after the removal of the drug. However, at the extended periods (greater than 24 h) after drug removal, RNA synthesis and cell growth resumed at the normal rate, and protein B23 relocated from nucleoplasm to nucleoli. This is in contrast to the results obtained from the experiments using higher doses (1 microgram/ml; 0.5 h) or longer (0.25 microgram/ml; 2 h) exposure of HeLa cells to actinomycin D, which induced irreversible B23 translocation as well as irreversible inhibition of cell growth and RNA synthesis. These results indicated that actinomycin D can be a reversible inhibitor depending on the drug extracellular concentrations and exposure times. Our results also indicated that "B23 translocation" is closely associated with states of cell growth and inhibition of RNA synthesis. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth in response to antitumor therapy.

Down-regulation of nucleophosmin/B23 during retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells.

Human promyelocytic leukemia HL-60 cells were induced to undergo granulocytic differentiation by treatment with retinoic acid (RA, 10 microM, 1-5 days). The steady-state level of nucleophosmin/B23 mRNA decreased during the RA-induced differentiation. There was also decrease in the level of total cellular nucleophosmin/B23 protein during the RA-induced differentiation. Stabilization and nuclear run-on assays indicate that the decrease in nucleophosmin/B23 mRNA in RA-treated HL-60 cells was transcriptionally regulated. Unlike c-myc mRNA, there was virtually no decline of nucleophosmin/B23 mRNA during the growth arrest by serum-starvation. The decrease in nucleophosmin/B23 mRNA expression in HL-60 cells subsequent to retinoic acid treatment can thus be attributed to cellular differentiation rather than the growth arrest induced by RA. Nucleophosmin/B23 antisense oligomer treatment significantly potentiated RA-induced cellular differentiation. Results of this study suggest that nucleophosmin/B23 is one of the key elements in the down-regulation of nucleolar function for cellular differentiation.

Mortalization of human promyelocytic leukemia HL-60 cells to be more susceptible to sodium butyrate-induced apoptosis and inhibition of telomerase activity by down-regulation of nucleophosmin/B23.

Vanadate (10 microM), a potent inhibitor of tyrosine phosphatase, added simultaneously potentiated BuONa-induced (1 mM) apoptosis. The steady-state level of nucleophosmin/B23 mRNA and the total cellular nucleophosmin/B23 protein decreased during the BuONa/vanadate-induced apoptosis. Stabilization and promotor transcriptional activity assays indicate that the decrease in nucleophosmin/B23 mRNA in BuONa/vanadate-treated HL-60 cells was transcriptionally regulated. A decline in telomerase activity was observed in HL-60 cells treated with BuONa/vanadate for 24-96 h. There was virtually no decline of nucleophosmin/B23 mRNA nor the telomerase activities during the growth arrest by serum-starvation. The decrease in nucleophosmin/B23 mRNA expression and telomerase activity in HL-60 cells subsequent to BuONa/vanadate treatment can thus be attributed to cellular apoptosis rather than the growth arrest induced by BuONa/vanadate. Nucleophosmin/B23 antisense oligomer treatment significantly potentiated BuONa-induced apoptosis and inhibition of telomerase activity. Results of this study suggest that nucleophosmin/B23 is one of the key elements in the down-regulation of nucleolar function for cellular apoptosis and mortalization.

Identification and characterization of a hexameric form of nucleolar phosphoprotein B23.

Under native purification conditions, an oligomeric form (Mr = 230,000) and monomeric form (37,000) of protein B23 were purified by affinity chromatography. Both forms were identified by Western blot immunoassay and ELISA. The molecular weight of the oligomeric form of protein B23 was estimated to be 230,000 with a Stoke's radius and a sedimentation coefficient of 51 A and 10 S, respectively. The oligomer (230 kDa) of protein B23 was dissociated into monomers (37 kDa) by treatment with 7 M urea. Quantitation of the monomer by gel scanning densitometry indicated that the oligomeric form of protein B23 is a hexamer containing four alpha and two beta monomers (37 kDa). A trace amount of nucleic acids (amounting to less than 3% of the total mass) was detected in the affinity-purified oligomers of protein B23. Protein B23 may be a structural element which is involved in ribosome transport or assembly in the nucleus.

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis.

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.

Physicochemical and immunological properties of albumin-associated dialkyl-ether phosphatidylcholine liposomes.

Multilamellar and unilamellar liposomes were prepared from sn-3-dihexadecylphosphatidylcholine/cholesterol/dicetylphosphate (7:2:1) in the presence of bovine serum albumin. Liposome-associated bovine serum albumin was separated from free bovine serum albumin by Blue Sepharose CL-6B affinity column chromatography. The chromatographed fractions were analyzed for their protein and liposomal phosphorus contents. The recovered albumin-containing liposomes were characterized morphologically by electron microscopy on negatively stained preparations. These preparations showed vesicular organizations of multilamellar or unilamellar phospholipid bilayers depending on the method of preparation used in each case. An analysis of the particle size distribution indicated that the mean radius was 280 +/- 50 nm for the multilamellar bovine serum albumin-liposomes and 150 +/- 50 nm for the unilamellar preparations. The efficacy of unilamellar and multilamellar dialkyl-ether phosphatidylcholine liposomes in eliciting antibody formation was examined. Mice were injected with liposome-entrapped bovine serum albumin and the albumin-specific plaque-forming cell response was evaluated. The unilamellar vesicles were found to be more effective than their multilamellar counterparts in promoting the elicitation of the anti-bovine serum albumin plaque-forming cell response. Within each category of lamellar structure, i.e., unilamellar or multilamellar bilayers, liposomes composed of dialkyl-ether phosphatidylcholines are less efficient than those of diacyl-ester phosphatidylcholines in potentiating the humoral immune response. These results demonstrate that liposome-mediated enhancement of the antibody response is determined, at least in part, by the lamellar arrangement of the vesicles and by the characteristic chemical structures of the phospholipids used.

Schedule-dependent sphinganine potentiation of retinoic acid-induced differentiation, cell growth inhibition, and nucleophosmin translocation in a human leukemia cell line (HL-60).

Induction of differentiation, inhibition of cell growth, and localization of nucleophosmin in HL-60 cells under the treatment of retinoic acid (RA) were studied. Bright nucleolar fluorescence was observed in control promyelocytic growing cells. The addition of RA in the culture system resulted in time- and dose-dependent induction of differentiation, cell growth inhibition, and nucleophosmin translocation from nucleoli to nucleoplasm. Unlike the control cells, many fewer nucleophosmin-associated preribosomal ribonucleoprotein particles (pre-rRNPs) could be obtained from nucleoli of RA-treated cells. Addition of sphinganine, an inhibitor of protein kinase C, facilitated the RA-induced differentiation, nucleophosmin translocation, and cell growth inhibition. Cells treated with sphinganine were more responsive to RA. Differentiation, translocation of nucleophosmin, and inhibition of cell growth occurred with lesser doses of RA or in shorter incubation times in the presence of sphinganine. Significant numbers of HL-60 cells could be rescued from the effects of RA upon the removal of RA after 2-h drug exposure. Pretreatment but not posttreatment of HL-60 cells with sphinganine, however, modulated the reversibility of the effects induced by short-exposure RA treatment. These results indicated that RA therapy can be improved by the pretreatment or the concurrent use of a modulator of protein kinase C activity. Nucleophosmin translocation as observed by immunofluorescence may be a simple and rapid method for assessing inhibition of cellular growth in response to differentiation inducers such as RA in cancer chemotherapy.

Autophagic adaptation is associated with exercise-induced fibre-type shifting in skeletal muscle.

AIM: Acute exercise is known to activate autophagy in skeletal muscle. However, little is known about how basal autophagy in skeletal muscle adapts to chronic exercise. In the current study we aim to, firstly, examine whether long-term habitual exercise alters the basal autophagic signalling in plantaris muscle and, secondly, examine the association between autophagy and fibre-type shifting. METHODS: Adult female Sprague-Dawley rats aged 2 months were randomly assigned to control and exercise groups. Animals in exercise group were kept in cages equipped with free access running wheels to perform habitual exercise for 5 months. Animals in the control group were caged in the absence of running wheels. Animals were sacrificed after the 5-month experimental period. Plantaris muscle tissues were harvested for analysis. RESULTS: We showed that long-term habitual exercise enhanced basal autophagy, but without altering expressions of autophagy proteins in plantaris muscle. Interestingly, sirtuin protein, a possible regulator of autophagy, was upregulated in plantaris muscle. Furthermore, we suspected that different types of muscle fibre adapted to chronic exercise in different ways. Long-term habitual exercise resulted in fibre-type shifting from type IIX to IIA in both gastrocnemius muscle and plantaris muscle. Intriguingly, our analysis demonstrated that LC3-II protein abundance is positively correlated with the proportion of type IIA fibre whereas it was negatively correlated with the proportion of type IIX fibre in plantaris muscle. PGC-1α protein abundance was positively associated with the proportion of type IIA fibre and LC3-II in plantaris muscle. CONCLUSION: These results suggest that basal autophagy is enhanced in plantaris muscle after long-term habitual exercise and associated with fibre-type shifting.

Cell cycle phase-dependent effect of retinoic acid on the induction of granulocytic differentiation in HL-60 promyelocytic leukemia cells. Evidence for sphinganine potentiation of retinoic acid-induced differentiation.

The efficiency of retinoic acid (RA)-induced differentiation was dependent on the position of HL-60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA-induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA-induced differentiation.

Effects of okadaic acid and vanadate on TPA-induced monocytic differentiation in human promyelocytic leukemia cell line HL-60.

Treatment of HL-60 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1-5 nM) induced inhibition of cell growth and the appearance of an adherent monocyte-like cell type in a dose- and time-dependent manner. The extent of TPA-induced monocytic differentiation was found to be markedly reduced by okadaic acid (OA) (35 nM). OA had to be present for the early 12 h during treatment with TPA to reduce the induction of monocytic differentiation. The majority of cells (80%) were non-adherent but morphologically resembled mature myelocytes or granulocytes after treatment with TPA (5 nM) in the presence of OA (35 nM). Vanadate (VD), on the other hand, enhanced the extent of monocytic differentiation induced by low-dose of TPA (1 nM). These results indicated that dephosphorylation by tyrosine protein phosphatase and serine-threonine protein phosphatase may play an important role in the induction of monocytic and granulocytic differentiation.

In vivo interaction of nucleophosmin/B23 and protein C23 during cell cycle progression in HeLa cells.

By using the cross-linking reagent, DSP, efforts were made to identify the protein(s) that interact with nucleophosmin/B23. A cross-linked protein complex at molecular weight of about 140 kDa was recognized by both nucleophosmin/B23 and protein C23 MAbs. Both C23 and nucleophosmin/B23 could be detected from the cross-linked complex immunoprecipitated by C23 MAb. The association between nucleophosmin/B23 and protein C23 while being observed at interphase and cytokinesis, was not detected in prometaphase and metaphase cells. Interactions of nucleophosmin/B23 with C23 not only could be found in cells in which nucleophosmin/B23 and C23 were both mainly localized to the nucleolus, but also in cells in which nucleophosmin/B23 and C23 had translocated from the nucleolus to the nucleoplasm during actinomycin D-induced cell growth inhibition. The purified recombinant GST-B23 being phosphorylated by prometaphase cell extracts (nocodazole-arrested cells) or cdc2 kinase could still be co-immunoprecipitated with C23. Consequently, the fact that nucleophosmin/B23 did not interact with C23 during mitosis could not be explained simply by mitotic phosphorylation of nucleophosmin/B23. Our findings suggest some possibilities for further elucidation of the actions of nucleophosmin/B23 and protein C23 in cell cycle progression and cell growth.

Modulation of the reversibility of actinomycin D cytotoxicity in HeLa cells by verapamil.

Actinomycin D treatment (0.001-0.005 micrograms/ml; 0.5-24 h) induced a dose and time response shifting of nucleolar to nuclear fluorescence. In the presence of verapamil, cells were more responsive to actinomycin D. Translocation of protein B23 occurred with lower doses of actinomycin D and in shorter incubation times in the presence of verapamil. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.05-0.25 micrograms/ml) induced 'reversible' translocation of protein B23 as well as 'reversible' inhibition of cell growth and RNA synthesis. Verapamil (5 microM) included in the cell culture after removal of actinomycin D inhibited the recoveries of cell growth, RNA synthesis as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. These results indicate that verapamil can potentiate the antiproliferating activity of actinomycin D by inhibiting reversibility of its cytotoxicity and suggest clinical application.

Different kinases phosphorylate nucleophosmin/B23 at different sites during G(2) and M phases of the cell cycle.

The recombinant GST-nucleophosmin/B23 and the truncated mutants were tested for phosphorylation in cell-free extracts of G(2) and M phases or by purified kinases. Our results indicated that a threonine residue at amino acids (a.a.) 185-240 was phosphorylated by cdc2 kinase during the entry of mitosis while the serine phosphorylation site at the middle acidic portion of the molecule (a. a. 83-152) was phosphorylated by casein kinase II during G(2) phase. Our results also showed that there was possibly another serine phosphorylation at site other than the middle portion of nucleophosmin/B23 (a.a. 83-152) during the entry of cells into mitosis. The demonstration of the characteristic changes in phosphorylation of nucleophosmin/B23 during the cell cycle implicates important role of nucleophosmin/B23 in the control of the fate of nucleoli and cell growth.

Berberine complexes with DNA in the berberine-induced apoptosis in human leukemic HL-60 cells.

Berberine, an alkaloid initially isolated from Chinese herbal medicine exhibited the ability to induce morphological changes and internucleosomal DNA fragmentation, characteristic of apoptosis in promyelocytic leukemia HL-60 cells. Cell cycle studies showed that only about 20% of the cells underwent apoptosis at the early time (6 h) of berberine (25 micrograms/ml) treatment; these appeared to be cells in S phase at the time of berberine treatment. At extended time (6-48 h), cells were cell cycle arrested, the number of cells of each phase, particularly the cells of S phase decreased and much more (> 50%) of the cells appeared with DNA content less than G1. Attempts were also made to isolate possible berberine-DNA complexes from cell cultures treated with berberine (25 micrograms/ml; 2-24 h). Shifts of absorption maxima of berberine in the direction of longer wavelengths were observed in the isolated berberine-DNA complexes. Palmatine, an analog of berberine, which was not able to induce apoptosis, also complexed with DNA in cells treated with palmatine (25 micrograms/ml; 2-24 h). Our results suggest that some important cellular processes other than the intracellular DNA-interacting action of berberine may be involved in the berberine-induced apoptosis in HL-60 cells.

Intracellular ATP is required for actinomycin D-induced apoptotic cell death in HeLa cells.

In the present report, we demonstrate that reduction of cellular ATP content with antimycin A blocks actinomycin D-induced apoptotic cell death in HeLa cells. Compared to cells (approximately 80%) treated with actinomycin D (1 microgram/ml; 48 h) alone in glucose-containing medium, a much smaller percentage of cells (approximately 20%) treated with actinomycin D in the presence of antimycin A in glucose-free medium shows morphological characteristic of apoptosis. ATP-depleted cells with or without actinomycin D treatment, on the other hand, die necrotically. In cells under actinomycin D short exposure treatment (1 microgram/ml; 1 h), apoptosis occurs when cellular ATP content is rapidly recovered after the removal of antimycin A and resupplementation of glucose-containing medium. In the incubation of isolated Triton-permeabilized cells with ATP ( > 0.5 mM), apoptotic nuclei become abundant 4 h after ATP treatment. These results implicate the requirement of ATP for the induction of apoptosis.

Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1).

Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4'-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC50 values of 9.5 +/- 3.2, 59.1 +/- 19.6 and 1423.3 +/- 212.2 ng/ml respectively. No inhibition of [3H]uridine incorporation was observed using actinomycin 4-4'-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 values for inhibition of [3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 "translocation" from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75-85% inhibition, only a uniform nucleoplasmic fluorescence was observed.