[A sampling survey of intraoperative facial nerve monitoring in China].

Objective: This study aims to investigate the current application and the level of knowledge of intraoperative facial nerve monitoring among medical staff in China. Methods: A comprehensive online questionnaire was conducted among medical professionals across different regions in China from October 2022 to February 2023. The survey exclusively targeted departments specializing in otolaryngology, head and neck surgery, neurosurgery, and oral and maxillofacial surgery. The questionnaire covered various aspects including general information, intraoperative facial nerve monitoring practices, training history, indications for monitoring, parameters used during monitoring procedures, as well as factors influencing its implementation. Results: A total of 417 participants from 31 provincial, municipal, and autonomous regions were included. Intraoperative facial nerve monitoring was found to be implemented in 227 (54.4%,227/417) repondents of 53 institutions (24.9%, 53/213). The top three indications for implementing this technique were acoustic neuroma, parotid gland surgery, and modified middle ear surgery (mastoidectomy). Herein 81.1%(184/227) medical staff involved in intraoperative facial nerve monitoring had received relevant training, 57.3%(130/227)-92.1%(209/227) reported a lack of clear description regarding recording thresholds, stimulation currents/frequencies/wave widths. Conclusion: The majority of the institutions surveyed have not yet adopted intraoperative facial nerve monitoring. Furthermore, significant gaps concerning the procedure exist. It is imperative to establish standards or guidelines to promote its better development and application.

Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells.

Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl-2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspase-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

[Effects of differentiation status on apoptosis of human leukemia HL60 cells].

The effects of differentiation of human leukemia HL60 cells on harringtonine(Har) and camptothecin(Cam) induced apoptosis(in these cells) were studied. When treated with phorbol 12-myriate, 13-acetate 16 nmol.L-1 for 24 h, the HL60 cells differentiated into monocyte/macrophage cells and were arrested at G1 phase. The differentiated cells were shown to be resistant to the Har and Cam induced apoptosis, but showed no change of expression of c-myc gene. HL60 cells incubated in 1.4% dimethyl sulfoxide for 48 h differentiated into granulocyte cells and were also gene arrested at G1 phase. The differentiated cells became resistant to the apoptosis induced by Cam, but not that by Har, and expression of c-myc decreased drastically in the differentiated cells. The results indicate that the differentiated status of human leukemia HL60 cells apparently affected the apoptosis induced by harringtonine and camptothecin, but it was irrelevant to the change of the expression of c-myc gene.

Cyclosporine inhibited calcium-mediated apoptosis of HL-60 cells.

AIM: To study the effects of cyclosporine (Cyc) on apoptosis of HL-60 cells. METHODS: Apoptotic cells induced by harringtonine (Har), camptothecin (Cam), or calcimycin (Cal), thapsigargin (Tha) were identified with DNA electrophoresis, morphology, and flow cytometry. Relative [Ca2+]i alteration of apoptotic HL-60 cells were determined with flow cytometry. RESULTS: Cal 1 mg.L-1 or Tha 0.5 mg.L-1 induced apoptosis of HL-60 cells. This effect was inhibited by nontoxic concentration of Cyc 1 mg.L-1. Cyc did not inhibit Har- or Cam-induced apoptosis of HL-60 cells. Both Cal and Tha increased intracellular calcium, whereas Har or Cam did not. CONCLUSION: Cyc inhibited apoptosis only induced by calcium increasement in HL-60 cells. The mechanism of apoptosis induced by Cal or Tha was different from that by Har or Cam.

Staurosporine blocked normal cells at G1/S boundary.

AIM: To reveal the regulating difference of G1/S-phase transition between normal and tumor cells by using staurosporine, an unspecific kinase inhibitor. METHODS: Flow cytometry, Dot blot, kinase activity assay, and electrophoresis. RESULTS: A 18-h treatment with staurosporine (5 micrograms.L-1) blocked normal cell line 2BS cells (normal human embryonic lung fibroblast, 5-20 passages) in G1 phase, decreased their thymidine kinase (TK) mRNA level and activity, and also dephosphorylated an intracellular 107 kDa protein. Meanwhile, all these effects in 2BS cells disappeared only by washing staurosporine away. Such kind of effects did not occur in tumor cell line BGC-823 cells (human stomach cancer cell). CONCLUSION: During the period of G1/S-phase transition, the kinases involved are more sensitive to staurosporine in normal cells than in tumor cells.

Inhibition of harringtonine-induced apoptosis by tetradecanoylphorbol acetate in human leukemia HL-60 cells.

AIM: To study the changes of the apoptosis induced by camptothecin (Cam) or harringtonine (Har) in human leukemia HL-60 cells after the cells were preincubated with tetradecanoylphorbol acetate (TA). METHODS: Chromatin condensation observation, flow cytometry, DNA agarose gel electrophoresis, and Dot blot hybridization. RESULTS: After the HL-60 cells were preincubated with TA 200 nmol.L-1 for 6 h, the apoptosis induced by Har 0.1 mg.L-1 for 3 h was drastically inhibited, and the apoptosis by Cam 0.2 mg.L-1 for 3 h was partly inhibited. On the other hand, the expression level of c-myc gene in HL-60 cells decreased apparently after the preincubation of TA. CONCLUSION: TA preincubation inhibited the apoptosis induced by Har obviously or by Cam partly in human leukemia HL-60 cells, and the expression of c-myc gene decreased drastically in the preincubated cells, which might result in the inhibition of apoptosis.

Resistance to apoptosis of harringtonine-resistant HL60 cells induced by tetrandrine.

AIM: To study the mechanism of resistance to apoptosis in the harringtonine (Har)-resistant HL60 cells with tetrandrine (Tet). METHODS: Growth inhibition, flow cytometry, DNA agarose gel electrophoresis, protein phosphorylation, and RNA dot hybridization. RESULTS: The resistant cells had no cross resistance to Tet. Tet induced the sensitive but not the Har-resistant HL60 cells to apoptosis. The high phosphorylation of protein < 30 kDa occurred when the resistant cells were treated with Tet. Tet and Har increased the expression of c-myc mRNA in the sensitive HL60 cells. The expression of c-myc mRNA in the resistant cells was obviously decreased and almost not changed in treatment with Tet and Har. CONCLUSION: Tet induced the sensitive but not the Har-resistant HL60 cells to apoptosis, and the resistance to apoptosis induced by Tet was associated with the high protein phosphorylation and reduction of the expression of c-myc mRNA.

[Apoptosis of HL-60 cells induced by Harringtonine: membrane blebs, nucleus blebs and chromatin condensation].

Using Video Enhancement Contrast (VEC) microscopy, we recorded the morphological changes of same HL-60 cell in the processes of apoptosis induced by harringtonine. Our results show that all of apoptotic cells need several nucleus blebs before their chromatin condensation. Every nucleus bleb is induced by a relative membrane bleb. The number of membrane blebs is much higher than that of nucleus blebs, so there are only some of membrane blebs which can induce nucleus blebs. It suggested that membrane and nucleus blebs probably are related to apoptotic chromatin condensation. After HL-60 cells pretreated with cytochalasin B(CB), apoptotic chromatin condensation delayed eight hours, but no membrane bleb, nucleus bleb and apoptotic body formed eventually. So membrane and nucleus blebs during apoptosis are related to microfilament re-organization and can accelerate apoptotic chromatin condensation, but are unnecessary for apoptotic chromatin condensation. All this suggested that nuclear changes and cytoplasmic changes during HL-60 cell apoptosis are independent.

Characteristics of harringtonine-resistant human leukemia HL60 cell.

AIM: To study the mechanisms of the resistance to harringtonine (Har) in the HL60 cells. METHODS: Growth inhibition, karyotype analysis, flow cytometry, Western blotting and polymerase chain reaction. RESULTS: The Har-resistant HL60 cell line, named HR20, showed cross resistance to homoharringtonine, doxorubicin, daunorubicin, vincristine, and colchicine. The growth doubling time and the cell numbers in G1 phase were increased. The accumulation of cellular daunorubicin in the resistant cells was obviously reduced, but distinctly increased by tetrandrine and verapamil. The numbers of telocentromeric chromosome increased and the chromosomal aberration more occured in the resistant cells. The resistant cells overexpressed multidrug resistant mdr-1 gene and P-glycoprotein 150 kDa. CONCLUSION: The Har-resistant HL60 cell strain belonged to a multidrug resistance strain, overexpressing mdr-1 gene and P-glycoprotein.

Correlation between expression of mdr-1 gene and oncogenes in human promyelocytic leukemic HL60 cell line and sublines.

AIM: To study the relationship between expression of oncogenes and multiple drug resistant (MDR) phenotype. METHODS: The drug resistant level of HL60 cell line and its sublines were determined with flow cytometry. RNA Dot blot hybridization was used to identify the expression of oncogenes and mdr-1 gene. RESULTS: The expression of mdr-1 gene was in the opposite relation with c-myc expression, but in the positive relation with c-H-ras gene expression in the multiple drug resistant cell lines. In non-MDR cell line HL60/RA, the expression levels of mdr-1, c-myc, and c-H-ras were the same as HL60 parental cells. CONCLUSION: Multiple drug resistance is related to not only mdr-1 expression, but also some oncogenes expression level.

[Changes of intracellular calcium, calmodulin in normal and tumor cells traggered by staurosporine].

Treated with low dosage staurosporine, the normal cell 2 BS were arrested in G 1 phase, but tumor cell BGC-823 were not. We measured the intracellular calcium, calmodulin, Ca(2+)-activited calmodulin (Ca(2+)-CaM) contents of single cells according to cell cycle with microphotometry. Our results showed that treatment of 5 ng/ml staurosporine for 18 h caused the changes of calcium and calmodulin. In 2 BS cells, CaM leval decreased in G 1 and S phase. In each cell cycle phase of BGC-823 cells, there was no changes in CaM level, but Ca(2+)-CaM level increased. The causes of that staurosporine blocked 2 BS cells at G 1 phase but not effected on cell cycle progression in BGC-823 cells may be the results of staurosporine decreased the contents of CaM at G 1 phase and inhibited the phosphoralation of p107 in 2 BS cells.

[Protein kinase inhibitor staurosporine enhances cytotoxicity of antitumor drugs to cancer cells].

Treated with low dosage (5 ng.ml-1) of staurosporine for 18 h, human embryo lung 2BS cells were blocked at the G1/S boundary, but human gastric carcinoma BGC-823 cells still kept their cell cycle. In comparison with IC50 of 2BS and BGC-823 cells treated with cell cycle phase specific antitumor drugs adriamycin, Ara-C and BLM A5 alone or combined with staurosporine 5 ng.ml-1, the IC50 values increased from 0.325 microgram.ml-1, 5 micrograms.ml-1 and 6.5 micrograms.ml-1 to 0.45 microgram.ml-1, 10 micrograms.ml-1 and 6.5 micrograms.ml-1, respectively in 2BS cells; but decreased from 0.325 microgram.ml-1, 25 micrograms.ml-1 and 1.1 micrograms.ml-1 to 0.07 microgram.ml-1, 6.25 micrograms.ml-1 and 0.4 microgram.ml-1, respectively in BGC-823 cells. These results suggest that combination of staurosporine 5 ng.ml-1 with antitumor drugs showed different effects on tumor cells and normal cells. With the GSH fluorescent probe mBCL, we found that GSH contents increased in 2BS cells treated with staurosporine 5 ng.ml-1.

[Retardation of human drug-resistant HL-60 cell in G1 phase and induction of sensitive cell to apoptosis by cyclosporine A].

To further study the relationship between resistance to apoptosis and drug resistance in harringtonine-resistant HL-60 cells (HR20), cyclosporine A (CsA) 20, 10 micrograms.ml-1 was shown to induce the sensitive HL-60 cells to apoptosis, showing a typical DNA "ladder" band. But the same concentrations of CsA retarded the HR20 cells in G1 phase and could not induce the cells to apoptosis. The cellular daunorubicin accumulation increased when HR20 cells were treated with low concentration of CsA and the reversal of drug resistance by CsA was unrelated to the retardation of cell cycle progression. High phosphorylation of about 50 kDa protein occured when HR20 cells were treated with CsA 10 micrograms.ml-1. The results domonstrate that cyclosporine A retarded the harringtonine-resistant HL-60 cells in G1 phase but induced HL-60 cells to apoptosis, and the retardation was unrelated to drug resistance.

[Induction of expression of MDR 1 gene by retinoic acid and DMSO and effects on rhodamine-123 efflux in HL-60 cell lines and resistant sublines].

Using dot blot hybridization and flowcytometry, the effects of differentiation inducers retinoic acid (RA) and dimethyl sulfoxide (DMSO) on the resistant level of HL-60 cells and its resistant subline cells were studied. When the cells were treated with RA 1 mumol.L-1 for 24 h, the expression of MDR 1 mRNA evidently increased in both HL-60 and its multidrug resistant subline cells. The efflux of Rho-123 in the multidrug resistant subline cells was slightly decreased. But, when the cells were treated with 2% DMSO for 24 h the efflux of Rho-123 increased obviously. The results suggest that RA can induce the expression of MDR1 gene but perhaps inhibit the function of pump glycoprotein 170 (Pgp-170) through phosphorylation/dephosphorylation pathway. However, DMSO could induce the expression of full function of Pgp.

[Drug resistance of doxorubicin-resistant CHO cell line].

Some characteristics of doxorubicin-resistant CHO cell line (RC1) were studied by means of cell biological methods and SDS-PAGE electrophoresis. The resistance factor was 16.5-fold, and RC1 revealed cross-resistances to colchicine, actinomycin and harringtonine. By indirect immunofluorescence assay, P-glycoprotein was not detected. Compared with CHO, the doxorubicin (Dox) uptake and accumulation of RC1 decreased, but the membrane fluidity of RC1 increased. The reduction in drug accumulation was correlated with increase in membrane fluidity. Dox was mainly distributed in the cell nucleus of CHO, but in both cytoplasm and nucleus of RC1. This suggested that Dox was transported more slowly in RC1 cytoplasm than in CHO cytoplasm, resulting in less Dox entrance into the cell nucleus of RC1 than into that of CHO. We also found that a 30-40 kDa nuclear protein which was expressed normally in CHO disappeared in RC1.

[Apoptosis resistance and its reversal in harringtonine resistant cell line].

Harringtonine (HT), a domestic antitumor drug extracted from Cephalotaxus hainanensis Li showed high chemotherapeutic efficacy on human acute granulocytic leukemia and acute myelocytic leukemia in clinics. Apoptosis of HL-60 cells can be induced by HT effectively; but for cells resistant to harringtonine, apoptosis can not be induced, even if the drug (HT) concentration is over 100 times of IC50 value. Although apoptosis occurred when its multidrugs resistance had been reversed by verapamil, compared with sensitive HL-60 cells, the time at which apoptosis happened delayed and the drug dosage increased. All these suggest that apoptotic resistance might be one of the marks of drug resistance in tumor cells, and apoptosis related factors could play a role in the formation of multidrug resistance.

Increase of calcium levels at interphase in NIH 3T3 cells.

The fluorescent calcium ion indicator dye Fluo-3 and DNA-binding dye Hoechst 33342 were employed to determine, in a quantitative microspectrofluorometric study, the intracellular calcium ion concentration ([Ca2+]i) and the DNA content of individual living NIH3T3 cells. The well-separated excitation and emission properties of these dyes allowed us to establish for each cell both the phase of the cell cycle using DNA content and [Ca2+]i. We found that the transition from G1, through S, to the G2 phase is accompanied by a two-fold increase in [Ca2+]i. The [Ca2+]i was inhomologous in each phase of the interphase (G1, S and G2) although [Ca2+]i in the S and G2 phases was never lower than certain threshold values in the G1 and S phases respectively. [Ca2+]i in G0 cells was lower than that in G1 cells. These changes in [Ca2+]i suggest that [Ca2+]i may be an important regulator of cell cycle progression.

[Studies on effective reversal of adriamycin-resistant Chinese hamster ovary cell line by verapamil].

A noncytotoxic dose of verapamil (Ver) 3 micrograms/ml was found to potentiate 10-fold the growth-inhibitory effects of adriamycin (ADM) in ADM-resistant Chinese hamster ovary cell line (RC1). Ver 3 micrograms/ml also reduced the IC50 value of ADM from 1.2 micrograms/ml to 0.08 microgram/ml in RC1 in clonogenic assay. The index of reversing resistance was 15-fold. In an attempt to elucidate the mechanism of the reversion of the multidrug resistance by Ver, Ver in combination with ADM was found to enhance intracellular ADM accumulation in RC1, and brought about more toxicity in RC1 than ADM alone; and Ver in combination with ADM was shown to retard RC1 in G2 + M phase by flow cytometry analysis.

Reversal of doxorubicin resistance by tetrandrine in Chinese hamster ovary cell line.

Tetrandrine (Tet) 0.5 microgram.ml-1 and 1 microgram.ml-1 potentiated 2.88- and 4.3-fold growth-inhibitory effects of doxorubicin (Dox) in Chinese hamster ovary cell line (CHO), respectively, while Tet 1 microgram.ml-1 and 2.5 micrograms.ml-1 potentiated 7.3- and 8.4-fold in its resistant cell line (CHO/Dox), respectively. The colony-forming efficiencies were reduced in CHO and CHO/Dox when the cells were treated with noncytotoxic doses of Tet 2.5 micrograms.ml-1 and 5 micrograms.ml-1 in combination with different concentration of Dox. Increase in accumulation of Dox in CHO/Dox cells was shown by fluorometry. The result indicated that Tet reversed the resistance to Dox in CHO/Dox cells.

[Enhancement of antitumor activity of harringtonine in human leukemia-60 cells in vitro by verapamil].

The effect of harringtonine (Har) alone and in combination with verapamil (Ver) on the proliferation of human leukemia-60 (HL-60) cells in vitro were studied. IC50 of Har alone to the cells was about 49 ng.ml-1 which was reduced to its 1/3.3 and 1/4.5 when used with Ver 1 and 2 micrograms.ml-1, respectively. In colony forming test, the survival fraction of the HL-60 cells treated with Har 15 and 30 ng.ml-1 plus Ver 2 micrograms.ml-1 was reduced to 1/3.3 and 1/8 of the cells as when treated with Har alone, respectively. The results suggested that Ver enhanced the antitumor activity of Har in vitro and may used as an enhancer of Har in vivo.