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.

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.

Acylated and unacylated ghrelin inhibit doxorubicin-induced apoptosis in skeletal muscle.

AIM: Doxorubicin, a potent chemotherapeutic drug, has been demonstrated previously as an inducer of apoptosis in muscle cells. Extensive induction of apoptosis may cause excessive loss of muscle cells and subsequent functional decline in skeletal muscle. This study examined the effects of acylated ghrelin, a potential agent for treating cancer cachexia, on inhibiting apoptotic signalling in doxorubicin-treated skeletal muscle. Unacylated ghrelin, a form of ghrelin that does not bind to GHSR-1a, is also employed in this study to examine the GHSR-1a signalling dependency of the effects of ghrelin. METHODS: Adult C57BL/6 mice were randomly assigned to saline control (CON), doxorubicin (DOX), doxorubicin with treatment of acylated ghrelin (DOX+Acylated Ghrelin) and doxorubicin with treatment of unacylated ghrelin (DOX+Unacylated Ghrelin). Mice in all groups that involved DOX were intraperitoneally injected with 15 mg of doxorubicin per kg body weight, whereas mice in CON group received saline as placebo. Gastrocnemius muscle tissues were harvested after the experimental period for analysis. RESULTS: The elevation of apoptotic DNA fragmentation and number of TUNEL-positive nuclei were accompanied with the upregulation of Bax in muscle after exposure to doxorubicin, but all these changes were neither seen in the muscle treated with acylated ghrelin nor unacylated ghrelin after doxorubicin exposure. Protein abundances of autophagic markers including LC3 II-to-LC3 I ratio, Atg12-5 complex, Atg5 and Beclin-1 were not altered by doxorubicin but were upregulated by the treatment of either acylated or unacyated ghrelin. Histological analysis revealed that the amount of centronucleated myofibres was elevated in doxorubicin-treated muscle while muscle of others groups showed normal histology. CONCLUSIONS: Collectively, our data demonstrated that acylated ghrelin administration suppresses the doxorubicin-induced activation of apoptosis and enhances the cellular signalling of autophagy. The treatment of unacylated ghrelin has similar effects as acylated ghrelin on apoptotic and autophagic signalling, suggesting that the effects of ghrelin are probably mediated through a signalling pathway that is independent of GHSR-1a. These findings were consistent with the hypothesis that acylated ghrelin inhibits doxorubicin-induced upregulation of apoptosis in skeletal muscle while treatment of unacylated ghrelin can achieve similar effects as the treatment of acylated ghrelin. The inhibition of apoptosis and enhancement of autophagy induced by acylated and unacylated ghrelin might exert myoprotective effects on doxorubicin-induced toxicity in skeletal muscle.

Involvement of nucleophosmin/B23 in TPA-induced megakaryocytic differentiation of K562 cells.

Human myelogenous leukaemia K562 cells were induced to undergo megakaryocytic differentiation by treatment with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, 24-72 h). The steady-state level of nucleophosmin/B23 mRNA decreased during the TPA-induced differentiation. There was also decrease in the level of cellular nucleophosmin/B23 protein and appearance of its degraded product (25 kDa) during the TPA-induced differentiation. Furthermore, K562/B23 (wild type), K562/D1 (delta280-294) and K562/D2 (delta263-294) cells were less, while K562/D3 (delta244-294) cells were more responsive to TPA-induced differentiation as compared to K562/vector or parental K562 cells. Activation of the ERK/MAPK was observed in parental K562 cells upon TPA treatment (5 nM, 5-30 min). As compared to K562/vector cells, less activation of ERK/MAPK was observed in K562/D2 cells, while ERK/MAPK was highly activated in K562/D3 cells upon TPA treatment. Our results indicate that nucleophosmin/B23 plays an important role in TPA-induced differentiation of K562 cells and the amino acids 244-294 at C-terminal of nucleophosmin/B23 could be an important site for regulation of cellular response to differentiation.

The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication.

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.

Increased stability of nucleophosmin/B23 in anti-apoptotic effect of ras during serum deprivation.

We obtained evidence that increased stability of nucleophosmin/B23 is involved in antiapoptotic effect of ras during serum deprivation. Nucleophosmin/B23 in serum-deprived (0% serum) NIH-3T3 cells was found to be highly unstable with a half-life less than 4 h. In contrast, nucleophosmin/B23 in serum-deprived ras-transformed (RAS-3T3) cells was as stable as that in serum-supplemented NIH-3T3 or RAS-3T3 cells. Treatment of RAS-3T3 cells with nucleophosmin/B23 antisense oligomer significantly potentiated the apoptosis induced by serum deprivation. Much less caspase-3 activity was noted in the lysate derived from serum-deprived RAS-3T3 cells compared with that in the lysate of serum-deprived NIH-3T3 cells. Cell permeable caspase-3 inhibitor added in the medium blocked the decrease of nucleophosmin/B23 and apoptosis induced by serum deprivation in NIH-3T3 cells. The inhibitor, on the other hand, promoted significant decrease of nucleolin/C23 in NIH-3T3 cells during serum deprivation. Unlike nucleolin/C23, down-regulation of nucleophosmin/B23 was thus not proliferation-dependent but caspase-3- and apoptosis-dependent. Our results indicate important relationships among ras, nucleophosmin/B23, activation of caspase-3, and induction of apoptosis.

Over-expression of nucleophosmin/B23 decreases the susceptibility of human leukemia HL-60 cells to retinoic acid-induced differentiation and apoptosis.

Stable clones of HL-60 cells in which nucleophosmin/B23 was over-expressed were established. Less percentages (4-20%) of nucleophosmin/B23 over-expressed (pCR3-B23) cells exhibited the morphological characteristic of apoptosis as compared with control vector-transfected (pCR3) cells (6-53%) during the 10 microM RA treatment for 1-4 days. In flow cytometry analysis, a block in the G1 phase was noted in all the pCR3-B23 and pCR3 cells after 2 days of 10 microM RA treatment and continued to be observed at all times measured up to 6 days. Smaller peaks of apoptotic cells with less than G1 DNA content were observed in pCR3-B23 as compared with pCR3 cells after 4-6 days of 10 microM RA treatment. As measured by expressions of differentiation markers and the functional assessment of the ability to reduce nitroblue-tetrazolium, our results further showed that over-expression of nucleophosmin/B23 decreased the response of the cells to RA-induced differentiation. Less cleavage of PARP and in vitro caspase-3 activity were observed in PCR3-B23 cells as compared with pCR3 cells treated with 10 microM RA for 3-4 days. IRF-1 was induced after 6 hr of 10 microM RA treatment in the pCR3-B23 and pCR3 cells. Significantly more nucleophosmin/B23 was co-immunoprecipitated with IRF-1 from pCR3-B23 cells than from pCR3 cells during RA treatment (10 microM; 24 hr, 96 hr). The IRF-1 transcriptional activity was found to be attenuated in pCR3-B23 cells as compared with pCR3 cells during the treatment of cells with RA. Nucleophosmin/B23, through interacting with IRF-1, plays an important role in the control of the susceptibility of cells to RA-induced differentiation and apoptosis.

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.

Nucleophosmin/B23 regulates the susceptibility of human leukemia HL-60 cells to sodium butyrate-induced apoptosis and inhibition of telomerase activity.

Stable clones of HL-60 cells in which nucleophosmin/B23 was over-expressed or down-regulated were established. The nucleophosmin/B23 protein levels in nucleophosmin/B23 over-expressed (pCR3-B23) or down-regulated (pCR3-32B) cells during BuONa/vanadate-induced apoptosis were characterized as compared with control vector-transfected (pCR3) cells. Over-expression of nucleophosmin/B23 resulted in decreased susceptibility of the cells to BuONa/vanadate-induced apoptosis. The response to inhibition of telomerase activity under BuONa/vanadate treatment also decreased in nucleophosmin/B23 over-expressed (pCR3-B23) cells. On the other hand, down-regulation of nucleophosmin/B23 made the cells more susceptible to BuONa-induced apoptosis or inhibition of telomerase activity. More precisely, by serial dilutions of each extract, the telomerase activity of the cells without drug treatment was determined and was found to be higher in nucleophosmin/B23 over-expressed (pCR3-B23) cells and lower in nucleophosmin/B23 down-regulated (pCR3-32B) cells as compared with the control vector-transfected (pCR3) cells. Our results indicate that nucleophosmin/B23 plays a functional role in the control of cellular apoptosis and immortalization.

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.

Berberine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of nucleophosmin/B23 and telomerase activity.

The steady-state level of nucleophosmin/B23 mRNA decreased during berberine-induced (25 microg/ml, 24 to 96 hr) apoptosis of human leukemia HL-60 cells. A decline in telomerase activity was also observed in HL-60 cells treated with berberine. A stable clone of nucleophosmin/B23 overexpressed in HL-60 cells was selected and found to be less responsive to berberine-induced apoptosis. About 35% to 63% of control vector-transfected cells (pCR3) exhibited morphological characteristics of apoptosis, while about 8% to 45% of nucleophosmin/B23-over-expressed cells (pCR3-B23) became apoptotic after incubation with 15 microg/ml berberine for 48 to 96 hr. DNA extracted from pCR3 cells contained more fragmented DNA than pCR3-B23 cells during treatment with 15 microg/ml berberine for 24 to 48 hr. Our results indicate that berberine-induced apoptosis is associated with down-regulation of nucleophosmin/B23 and telomerase activity. We also suggest that nucleophosmin/B23 may play an important role in the control of the cellular response to apoptosis induction.

Down-regulation of nucleophosmin/B23 mRNA delays the entry of cells into mitosis.

In investigating the regulation of nucleophosmin/B23 mRNA expression at the entry of mitosis, the results of Northern gel blot analysis showed that the nucleophosmin/B23 mRNA levels significantly increased in prometaphase (nocodazole-arrested) or metaphase (colchicine-arrested) cells collected by mitotic shake-off. A higher level of nucleophosmin/B23 mRNA was detected in all the collected mitotic cells arrested by treatment with nocodazole for 10-18h as compared to that in G2 cells. An attempt was then made to determine whether the regulation of nucleophosmin/B23 mRNA plays a role in the control of entry into mitosis. Down-regulation of nucleophosmin/B23 mRNA by transfection of its antisense construct resulted in the delay of cells entering mitosis. The demonstration of the characteristic changes in the mRNA level of nucleophosmin/B23 during the entry of cells into mitosis implicates the importance of nucleophosmin/B23 in the control of the mitotic fate of nucleoli and cell growth.

Decrease in nucleophosmin/B23 mRNA and telomerase activity during indomethacin-induced apoptosis of gastric KATO-III cancer cells.

The mRNA expression of nucleophosmin/B23 in gastric cancers (T) and the matched adjacent "normal" gastric mucosa (N) obtained from patients without any preoperative treatment were determined. Telomerase activity was detected in tumor tissues from six of seven patients. Analysis of the adjacent "normal" gastric mucosa in the same patients revealed all seven were negative for telomerase activity. In comparing clinical data for all seven patients, the stages of cancer seemed to be associated with T/N nucleophosmin/B23 mRNA expression. Cancers of later stages seemed to have higher T/N nucleophosmin/B23 mRNA ratio. After 3-4 days of 1 mM indomethacin treatment about 60-85% of gastric cultured KATO III cancer cells exhibited the features with highly condensed nuclei and decrease in cell size. Concomitant with the increase in the percentage of KATO III cells exhibiting the morphological features of apoptosis, there was a decrease in the viability of cells as determined by exclusion of trypan blue. A decline in telomerase activity in indomethacin-treated versus untreated cells was observed over times (2-4 days). The steady-state level of nucleophosmin/B23 mRNA, as determined by the levels of radioactivity of the hybridizing bands also decreased during the indomethacin treatment. At some times after the removal of indomethacin, cell growth and telomerase activity resumed in little extent (approx. 60%). When nucleophosmin/B23 antisense oligonucleotide was included in the cell culture upon removal of indomethacin, virtually no recovery of cell growth and telomerase activity were observed.

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.

Evidence for the ability of nucleophosmin/B23 to bind ATP.

By taking advantage of its ability to be retained by ATP-agarose, we have demonstrated that nucleophosmin/B23 is capable of binding ATP. The specificity of the binding was confirmed by the absence of significant binding to AMP-agarose and by its loss when nucleophosmin/B23 in nuclear extracts was preincubated with ATP. Preincubation of the nuclear extracts with other ribonucleotide triphosphates (GTP, CTP, UTP) did not compete for the binding of nucleophosmin/B23 to ATP-agarose. The purified recombinant nucleophosmin/B23 was also able to be retained by ATP-agarose. The Kd for binding of ATP to the purified recombinant nucleophosmin/B23, on the basis of retention on a nitrocellulose membrane, was 86.5+/-8.3 microM; the number of binding sites was 0.68 per nucleophosmin/B23 protein molecule. To determine the possible ATP-binding site of nucleophosmin/B23, various deletion clones including the two mutants in which the putative ATP-binding sequence had been deleted were constructed. Deletion of the portions of the molecule (residues 83-152 and 185-240) had little effect on the ATP binding. The C-terminal deleted mutant (residue 242 to the C-terminus deleted) lost most of its ability to be retained by ATP-agarose and to bind [alpha-32P]ATP on a nitrocellulose membrane. The results indicate that the C-terminal portion (residues 242-294) contains the essential ATP-binding site of nucleophosmin/B23.

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.

Antiapoptotic effect of ras in the apoptosis induced by serum deprivation and exposure to actinomycin D.

Serum deprivation or exposure of NIH 3T3 cells to actinomycin D (0.25-1.0 microgram/ml; 1 h) was associated with the accumulation of numerous apoptotic cells, as identified by their condensed nuclei and the decrease in cell size. In contrasts, v-H-ras-transformed NIH 3T3 cells were found to be resistant to this apoptosis induction. When v-H-ras-transformed cells were first pretreated for 24 h with 50 microM mevastatin, an agent which is known to be capable to deactivate the ras function, cell viability decreased and apoptotic cells became abundant (approximately 60-80%) 72 h after serum deprivation or exposure to actinomycin D. During the serum deprivation of NIH 3T3 cells, appearance of the apoptotic cells was preceded by G1 phase arrest. Accumulation of cells in the G1 phase was also observed in v-H-ras-transformed cells 24 h after serum deprivation. At later times (48-72 h), v-H-ras-transformed cells seemed to be capable of breaking through the G1 arrest and were then found to be distributed normally in the cell cycle.

Dose-dependent effects of berberine on cell cycle pause and apoptosis in Balb/c 3T3 cells.

In determining the morphological appearance of Balb/c 3T3 cells from berberine-treated (100 and 200 micrograms/ml) cultures by light microscopy demonstrated that the high berberine concentration (200 micrograms/ml) treatment was associated with the accumulation of numerous apoptotic cells, as identified by condensed nuclei and decrease in cell size. On the other hand, accumulation of cells in G2/M phase instead of induction of apoptosis was observed after 48-72 h of 100 micrograms/ml berberine treatment. Berberine was found mainly in cytoplasm during berberine-induced (100 micrograms/ml) cell cycle G2/M arrest, while it was highly concentrated in nuclei in the induction of apoptosis under high dose of berberine (200 micrograms/ml) treatment. Further addition of berberine (100-200 micrograms/ml) had little effect on the induction of apoptosis in the cells that had already been exposed to 100 micrograms/ml of berberine for 48 h. Our results suggest that there may exist in Balb/c 3T3 cells an important threshold for regulation of cell cycle pause and induction of apoptosis, that is dose-dependent.

Decreased accumulation and dephosphorylation of the mitosis-specific form of nucleophosmin/B23 in staurosporine-induced chromosome decondensation.

Nucleophosmin/B23 is highly phosphorylated by cdc2 kinase during mitosis, and this phosphorylation most probably has a role in initiating and controlling the entry of cells into mitosis [Peter, Nakagawa, Doree, Labbe and Nigg (1990) Cell 60, 791-801]. In the present study, the protein kinase inhibitor staurosporine has been used to examine possible changes in nucleophosmin/B23 at mitosis in HeLa cells. Addition of staurosporine to HeLa cells already arrested at mitosis by nocodazole causes: (i) decreased accumulation of the mitosis-specific form of nucleophosmin/B23, (ii) dephosphorylation of nucleophosmin/ B23, (iii) redistribution of nucleophosmin/B23 to the cytosol, and (iv) concomitant decondensation of chromosomes. These results suggest that the mitosis-specific phosphorylated form of nucleophosmin/B23 may play a role in maintaining mitotic chromosomes in their condensed state.

Potentiation of sodium butyrate-induced apoptosis by vanadate in human promyelocytic leukemia cell line HL-60.

Vanadate (10 microM ), a potent inhibitor of tyrosine phosphatase, added simultaneously potentiated the sodium butyrate (BuONa)-induced growth inhibition. Furthermore, at 1 mM BuONa alone, after 96 h of incubation, about 20 +/- 5% of cells exhibited the morphological characteristic of apoptosis, as established by nuclear changes (condensed and fragmented nuclei) and decrease in cell size. After treatment of cells with 1 mM BuONa in the presence of 10 microM vanadate, apoptotic cells became more abundant; 90 +/- 3% of cells presented morphological characteristics of apoptosis after 96 h of incubation. Flow cytometric measurement of DNA content demonstrated the accumulation of cells in G1 phase after 72 h of incubation with 1 mM BuONa alone. In the presence of vanadate (10 microM ), accumulation of cells in G1 phase appeared after shorter times of incubation (48 h) with BuONa. A substantial increase in the proportion of cells with degraded DNA characteristic of apoptosis was observed after 48- to 72-h incubation with BuONa in the presence of vanadate. BuONa-induced apoptosis was accompanied by the increase of tyrosine phosphorylation of cellular proteins pp37 and pp97. Our results raised the possibility that regulation of tyrosine phosphorylation of pp37 and pp97 is an important event that heralds the BuONa-induced apoptosis.