The efficacy of the combination of venetoclax and hypomethylating agents versus HAG agents in patients with acute myeloid leukemia: a retrospective study.

OBJECTIVES: The purpose of this study was to compare the effectiveness of the combination of venetoclax and hypomethylating agents with the HAG regimen. METHODS: We studied 52 cases of newly diagnosed AML and 26 cases of relapsed refractory AML, (including AML patients with treatment-related and ELN-adverse risk disease (n = 50)). These patients were treated with venetoclax and hypomethylating agents and HAG regimens, respectively. RESULTS: Twenty-nine patients newly diagnosed with acute myeloid leukemia were treated with VEN-HMA (venetoclax-hypomethylating agent), while 23 patients were treated with HAG. The median age of the VEN-HMA group was 70 years, while the HAG group had a median age of 69 years. The VEN-HMA group achieved a significantly higher rate of complete remission (82.7%) compared to the cohort treated with the HAG regimen (21.7%) (P < 0.001). At the same time, the VEN-HMA group exhibited a significant survival advantage compared to the HAG treatment group(HR = 0.328, 95%CI: 0.158-0.683, P = 0.003).In patients with relapsed and refractory acute myeloid leukaemia, 43.8% of patients in the VEN-HMA treatment group achieved complete remission, which was similar to the 50% in the HAG treatment group (P > 0.99). The median overall survival was similar between the VEN-HMA and HAG groups, with 4 and 3.67 months, respectively (P = 0.290). CONCLUSIONS: In conclusion, our analyses indicated that VEN-HMA resulted in better therapeutic outcomes compared to HAG for newly diagnosed AML patients, with higher rates of complete remission and overall survival. In relapsed/refractory AML patients, there was no significant difference in the efficacy of the two treatments and further studies with larger sample sizes are warranted.

Carboxyl terminal activating region 3 of latent membrane protein 1 encoded by the Epstein­Barr virus regulates cell proliferation and protein expression in NP69 cells.

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein­Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232­351; amino acid residues including 232­351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69­LMP1Δ232­351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232­351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69­LMP1Δ232­351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2­fold change) in NP69­LMP1Δ232­351 cells compared with NP69­LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232­351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1­CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells.

Identification of a TRBD zinc finger-interacting protein in Giardia duodenalis and its regulation of telomerase.

BACKGROUND: Giardia duodenalis causes giardiasis, with diarrhea as the primary symptom. The trophozoite proliferation of this zoonotic parasite is mainly affected by telomerase, although the mechanism of telomerase regulation has not been thoroughly analyzed. METHODS: This study was performed to identify the telomerase RNA-binding domain (TRBD)-interacting protein in G. duodenalis and its regulation of telomerase. Interaction between TRBD and interacting proteins was verified via pulldown assays and co-immunoprecipitation (co-IP) techniques, and the subcellular localization of the protein interactions was determined in vivo via split SNAP-tag labeling. The hammerhead ribozyme was designed to deplete the mRNA of TRBD-interacting proteins. RESULTS: Using TRBD as bait, we identified zinc-finger domain (ZFD)-containing proteins and verified it via pulldown and co-IP experiments. Protein-protein interaction occurred in the nuclei of 293T cells and both nuclei of G. duodenalis. The hammerhead ribozyme depleted ZFD mRNA levels, which reduced the reproduction rate of G. duodenalis, telomerase activity and telomere length. CONCLUSIONS: Our findings suggest that ZFD may regulate telomere function in G. duodenalis nuclei.

[Values of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in predicting sensitivity to intravenous immunoglobulin in Kawasaki disease].

OBJECTIVE: To study the values of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) in predicting the sensitivity to intravenous immunoglobulin (IVIG) in Kawasaki disease (KD). METHODS: A retrospective cohort study was conducted in 404 children with newly diagnosed KD. The data on routine blood tests, NLR, and PLR were collected before and after IVIG treatment. The receiver operating characteristic (ROC) curve was used to determine the cut-off values of NLR and PLR in predicting the insensitivity to IVIG. A logistic regression analysis was used to identify independent predictive factors for insensitivity to IVIG. RESULTS: Of all patients, 31 were insensitive to IVIG. Compared with the IVIG sensitivity group, the IVIG insensitivity group had a significantly higher incidence rate of coronary artery ectasia (P<0.01), a shorter course of disease when IVIG therapy was initiated (P<0.05), and significantly higher NLR, PLR, and C-reactive protein (CRP) level before and after treatment (P<0.05). The optimal cut-off values for NLR and PLR to predict IVIG insensitivity were 4.36 and 162 before IVIG treatment and 1.45 and 196 after treatment. The multivariate regression analysis showed that the course of disease before IVIG treatment, CRP before IVIG treatment, and NLR and PLR before and after IVIG treatment were independent predictive factors for IVIG insensitivity. CONCLUSIONS: NLR and PLR can be used to predict IVIG insensitivity in children with KD.

PTEN regulates BCRP/ABCG2 and the side population through the PI3K/Akt pathway in chronic myeloid leukemia.

A small population of cancer stem cells named the "side population" (SP) has been demonstrated to be responsible for the persistence of many solid tumors. However, the role of the SP in leukemic pathogenesis remains controversial. The resistance of leukemic stem cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ATP-binding cassette sub-family G member 2 (ABCG2), is well known as a specific marker of the SP and could be controlled by several pathways, including the PI3K/Akt pathway. Our data demonstrated that compared with wild-type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated phosphatase and tensin homologue deleted on chromosome -10 (PTEN) and activated phosphorylated-Akt (p-Akt). PTEN and p-Akt downregulation could be abrogated by both the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin. Moreover, in CML patients in the accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression was accompanied by the loss of PTEN protein and the up-regulation of p-Akt expression. These results suggested that the expression of ABCG2 and the SP may be regulated by PTEN through the PI3K/Akt pathway, which would be a potentially effective strategy for targeting CML stem cells.

MiR-223 regulates human embryonic stem cell differentiation by targeting the IGF-1R/Akt signaling pathway.

Currently, there are difficulties associated with the culturing of pluripotent human embryonic stem cells (hESCs), and knowledge regarding their regulatory mechanisms is limited. MicroRNAs (miRNAs) regulate gene expression and have critical functions in stem cell self-renewal and differentiation. Moreover, fibroblast growth factor (FGF) and the insulin-like growth factor receptor (IGF-1R) are key activators of signaling in hESCs. Based on the identification of complementary binding sites in miR-223 and IGF-1R mRNA, it is proposed that miR-223 acts as a local regulator of IGF-1R. Therefore, levels of miR-223 were detected in differentiated versus undifferentiated hESCs. In addition, proliferation, apoptosis, and differentiation were assayed in these two hESC populations and were compared in the presence of exogenous miR-223 and miR-223 inhibitor. Inhibition of miR-223 was found to maintain the undifferentiated state of hESCs, while addition of miR-223 induced differentiation. Furthermore, these effects were found to be likely dependent on IGF-1R/Akt signaling.

Inactivation of PTEN increases ABCG2 expression and the side population through the PI3K/Akt pathway in adult acute leukemia.

Both the occurrence and recurrence of acute leukemia (AL) might suggest the presence of leukemia stem cells. Side population (SP) cells, exhibiting stem cell-like properties, express ABCG2 (breast cancer resistance protein [BCRP]). This study revealed that over-expression of ABCG2 in Jurkat and HL60 cells led to an increased SP fraction, up-regulated levels of phosphorylated-PI3K and phosphorylated-Akt, and enhanced drug resistance, all of which could be attenuated by treatment with either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin. ABCG2 expression and SP cell counts were further characterized in 222 adult AL patients at three disease stages: upon diagnosis, at remission and at refractory/relapse (R/R), while 10 healthy donors served as the normal controls. Only a small fraction of the ABCG2+population (0.05-12.3%) and SP cells (0.02-1.60%) were observed in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. In the normal control population, the SP cell fraction represented a statistically higher percentage of total cells compared to the fraction of SP cells upon diagnosis or relapse in both AML and ALL. In addition, we demonstrated that ABCG2 expression and SP cell ratios can be upregulated by the inactivation of phosphatase and tensin homolog (PTEN) protein, achieved in this study by removing inhibition of the PI3K/Akt pathway. Collectively, this study suggests that the PTEN/PI3K/Akt pathway up-regulates ABCG2 expression and the SP cell population and is a potential AL-specific treatment target worth investigating further.

MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells.

INTRODUCTION: Tissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells. METHODS: hESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis. RESULTS: We first found that TF was expressed only in trophoblasts and granulocyte-monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway. CONCLUSIONS: The miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.

[Effects of Epstein-Barr virus latent membrane protein 1 mediated by nuclear factor-kappaB on transformation and tumorigenesis of rat-1 cells].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an oncoprotein coded by EBV genome. This study was to investigate the effects of EBV LMP1 on transformation and tumorigenesis of Rat-1 cells. METHODS: Retrovirus plasmids pLNSX-LMP1 and pBabe-IkappaBalpha, constructed by gene recombination technique, were cotransfected respectively with nuclear factor-kappaB luciferase reporter (pNF-kappaB-luc) into 293 cells. The actions of LMP1 in activating NF-kappaB and IkappaBalpha in inhibiting NF-kappaB were measured by luciferase activity assay. Moreover, pLNSX-LMP1 and pBabe-IkappaBalpha were transfected respectively into the ecotropic retrovirus packaging cell line PA317 to generate LMP1 retrovirus (RV-LMP1) and IkappaBalpha retrovirus (RV-IkappaBalpha). After Rat-1 cells were infected by RV-LMP1 alone or RV-LMP1 combined RV-IkappaBalpha, their malignant transformation phenotype was detected by colony forming assay and nude mice tumorigenicity assay. RESULTS: When pLNSX-LMP1 and pBabe-IkappaB were cotransfected at a ratio of 1:1, IkappaBalpha inhibited LMP1-mediated NF-kappaB activation by 75%û and at a ratio of 3:1, it almost completely inhibited LMP1-mediated NF-kappaB activation. IkappaBalpha obviously inhibited LMP1-mediated malignant phenotype of Rat-1 cells:colony formation number on plates were significantly decreased from (368+/-7)/well and (287+/-17)/well to (59+/-6)/well and (8+/-2)/well (P<0.001). Foci in soft agarose were decreased from (477+/-13)/well and (347+/-10)/well to (61+/-15)/well and (95+/-7)/well (P<0.001). The ability of tumorigenicity in nude mice was markedly decreased: tumor volume was decreased from (1.61+/-0.23) cm3 to (0.20+/-0.08) cm3 (P<0.001). CONCLUSION: EBV-LMP1 could lead to transformation and tumorigenesis of Rat-1 cells by activating NF-kappaB.

[Mechanism of migration in CNE2 cells promoted by EBV-LMP1].

OBJECTIVE: To investigate the mechanism of migration phenotype change induced by EBV-LMP1 in nasopharyngeal carcinoma (NPC) cell line CNE2. METHODS: Retroviruses RV-LNSX, RV-LMP1, and RV-LMP1(TRADD) prepared previously were used to infect CNE2 cells. After selection with G418, the morphology, the ability of motion and migration in extracellular matrix, expression of LMP1 and E-Cadherin in transgenic cells were observed or detected. Meanwhile, pEcad-luc was respectively co-transfected with pLNSX, pLNSX-LMP1, and pLNSX-LMP1(TRADD), to examine the effect of LMP1 on the transcriptional activity of E-Cadherin promoter in 293 cells. RESULTS: Compared with CNE2 and CNE2-LNSX cells, CNE2-LMP1 cells morphologically changed from typical epithelial appearance to long-spindle fibroblastic morphology with the concomitant loss of cell-to-cell contact, and relative migration of CNE2-LMP1 cells obviously increased (n=3, P< 0.05), while the expression of E-Cadherin was negative in CNE2-LMP1 cells. The transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin was suppressed by LMP1, and the level of suppression was correlated with the concentration of pLNSX-LMP1 (0.2,0.6 and 1.0 microg). LMP1(TRADD) didn't induce the changes of morphology and migration phenotype, nor suppress the transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin in CNE2 cells. CONCLUSION: EBV-LMP1 promotes the migration and down-regulates the expression of E-Cadherin in CNE2 cells. The mechanism is that EBV-LMP1 suppresses the transcriptional activity of E-Cadherin promoter. TRADD of carboxyl terminus of LMP1 may be the main active domain to promote the migration in NPC cells.

Resveratrol prevents CsA inhibition of proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through an ER/NO/cGMP pathway.

The purpose of this study was to investigate the in vitro effects of resveratrol (RSVL) and cyclosporin A (CsA) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures. Application of RSVL (10(-8) -10(-6) mol l(-1)) resulted in a dose-dependent increase in [3H]-thymidine incorporation, alkaline phosphatase (ALP) activity and calcium deposition of BMSCs cultures, which was accompanied with the increase of NO production and cGMP content. Concurrent treatment with the estrogen receptor antagonist ICI182,780 (10(-7) mol l(-1)) or the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (6 x 10(-3) mol l(-1)) abolished the RSVL (10(-6) mol l(-1))-induced increase in NO production and cGMP content and eliminated the RSVL-induced increase in proliferation and osteoblastic differentiation of BMSCs. In contrast, CsA (10(-6) -10(-5) mol l(-1)) dose-dependently decreased [3H]-thymidine incorporation, ALP activity and calcium deposition of BMSCs cultures, which was accompanied with the reduction of NO production in the conditioned media. Concurrent treatment with RSVL (10(-6) mol l(-1)) significantly reversed the CsA (3 x 10(-6) mol l(-1))-mediated decrease in NO production and restored the proliferation and differentiation potential of BMSCs. Our data suggest that (1) the NO/cGMP pathway may play an important role in both RSVL-induced and CsA-inhibited proliferation and osteoblastic differentiation of mouse BMSCs, and (2) RSVL may act through an ER/NO/cGMP pathway to reverse the inhibitory effect of CsA on BMSC cultures. Taken together, the data suggest that RSVL may prevent osteoporosis induced by CsA.

[Analysis of differential proteins in laryngeal carcinoma cell line Hep-2 with transfection of LCRG1].

BACKGROUND & OBJECTIVE: Laryngeal carcinoma-related gene 1 (LCRG1), a novel laryngeal carcinoma-related tumor suppressor gene, was cloned with mRNA differential display method. Previous researches showed LCRG1 might inhibit cell growth, proliferation, colony formation in soft agar, and tumorigenesis of laryngeal carcinoma cell line Hep-2. This study was to screen the proteins associated with the tumor suppressive function of LCRG1 by comparative proteomics method. METHODS: The whole cellular proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were extracted and separated by two-dimensional gel electrophoresis (2-DE) technology. After electrophoresis, the gels were stained by Coomassie Brilliant Blue G-250, and analyzed using PDQuest software. The differentially expressed proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), or electrospray ionization-quadrupole time-of-flight MS/MS (ESI-Q-TOF MS/MS), and identified through searching database with Mascot software. RESULTS: The well-resolved, reproducible 2-DE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were established. The total protein spots were 1,075+/-43 in Hep-2/LCRG1 cells and 1,027+/-23 in Hep-2/pcDNA3.1(+) cells, with an average matching rate of 91%. Using mass spectrometry technology, 20 differential protein spots between the 2 cell lines were identified. Among them, 16 were identified by MALDI-TOF-MS, and 4 were identified by ESI-Q-TOF MS/MS. Some of the identified proteins were characterized as members of cellular transcription and metabolism enzymes. CONCLUSIONS: In this study, 2-DE gels of Hep-2/LCRG1 cell line with high expression of LCRG1 mRNA and vector control Hep-2/pcDNA3.1(+) cell line were established; some differential proteins related to LCRG1 were identified by mass spectrometry. These data will help to illustrate the molecular mechanism of LCRG1.

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture.

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.

[Molecular cloning and functional analysis of STGC3 - a novel gene on chromosome 3p21].

BACKGROUND & OBJECTIVE: A locus of loss of heterozygosity (LOH) with high frequency has been found on chromosome 3p21 in nasopharyngeal carcinoma (NPC). On the basis of our former research, this study was designed to clone and analyze a novel NPC-associated gene at this locus. METHODS: The full-length cDNA sequence of this gene was obtained by plasmid cDNA sequencing and RACE,and analyzed by bioinformatics. The pEGFP-C2/STGC3 fusion mammalian expression vector was constructed, and transfected into COS7 and CNE2 cell lines mediated by lipofectin to analyze the subcellular localization of gene expressing proteins. The expression of STGC3 was detected in normal tissues and tumor cell lines by Northern blot. RESULTS: An 1 271 bp full-length cDNA sequence of gene,which had no obvious homology with other known genes in bioinformatic databases,was obtained. This gene,named STGC3 (GenBank accession number:AY078383), localized on chromosome 3p21, and encodes a protein consisting of 146 amino acids. Protein localization analysis under fluorescent microscope indicated that STGC3 fusion protein was distributed in nucleus and cytoplasm 24-48 hours after transfection. MTE(TM)Array2 Northern blot analysis showed STGC3 expressed in both normal tissues and tumor cells,while its expression down-regulated in many tumor cell lines, such as Burkitt's lymphoma cell line Daudi. CONCLUSIONS: STGC3 is a novel gene, and down-regulated in NPC and many other tumor cell lines. STGC3 fusion protein distributes in cytoplasm and nucleus.

[Expression establishment and functional analysis of breast cancer resistance protein with doxycycline induced tet regulating system in mouse fibroblast cell line PA317].

BACKGROUND & OBJECTIVE: Breast cancer resistance protein (BCRP), discovered in 1998, is a novel member of ATP-binding cassette (ABC) membrane transporters superfamily. This study was to establish the functional expression of BCRP with doxycycline (Dox) induced Tet regulating system in mouse fibroblast cell line PA317, provide an ideal experimental platform for understanding the mechanism of BCRP-mediated drug resistance, and develop effective methods to reverse the drug-resistance. METHODS: Tet-on regulating plasmid was transferred into PA317 cells under the Dox induced Tet-on regulating system, and stable expression of Tet-on was established in PA317 cells through G418 selection. The response plasmid of recombinant pTRE-BCRP was transferred into positive PA317/Tet-on cells, and stable expression of BCRP was established through hygromycin selection. Six single cellular clones were taken and cultivated in amplification. Positive PA317/Tet-on/TRE-BCRP cells, which showed well dose-response in expression of BCRP with different concentration of Dox induction, were selected by both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The inhibitory effects of mitoxantrone on positive PA317/Tet-on/TRE-BCRP cells with various concentrations of Dox induction were detected by MTT method. Ko143, a specific inhibitor of BCRP, was used to detect the drug sensitivity of positive PA317/Tet-on/TRE-BCRP cells to mitoxantrone. Fluorescence intensity of remaining intracellular mitoxantrone in positive PA317/Tet-on/TRE-BCRP cells with different expression of BCRP was analyzed by flow cytometry (FCM). RESULTS: The positive No.5 PA317/Tet-on /TRE-BCRP cell clone showed well dose-response in expression of BCRP with different concentration of Dox induction; its drug-resistance against mitoxantrone has positive correlation with BCRP expression (r=0.995, P=0.002); its drug sensitivity to mitoxantrone was significantly enhanced by Ko143 (P< 0.05). Furthermore, different expression of BCRP induced by various concentrations of Dox caused different intracellular mitoxantrone retention in this cell clone. CONCLUSION: Functional expression of BCRP under Dox induced Tet regulating system was successfully established in PA317 cells, which provided an ideal experimental platform for further study of BCRP.

[Expression and structure of BNIP3L in lung cancer].

BACKGROUND & OBJECTIVE: Bcl-2/E1B 19kDa interacting protein3-like (BNIP3L) gene is a tumor suppressor gene cloned from a human fetal liver cDNA library, which is located at 8p21, one of the high frequent regions of loss of heterozygosity (LOH) in lung carcinoma. BNIP3L protein can interact with antiapoptotic proteins, such as Bcl-2, Bcl-x(L), E1B19K, which promotes apoptosis. This study was designed to explore the correlation of alteration of expression and structure of BNIP3L gene with the progression of lung cancer. METHODS: The expression and structure of BNIP3L gene in 4 lung cancer cell strains and 30 tissues were determined by SP immunohistochemistry, immunoblot, semi-quantitative reverse transcription-PCR (RT-PCR), PCR-single strain conformation polymorphism (PCR-SSCP). RESULTS: (1) In 4 lung cancer cell strains, BNIP3L protein was not detected in A549, NCI-H460, NCI-H446, except for NCI-H520, in which the protein expression level was slightly lower than that in immortal bronchial epithelial cell strain HBE4-E6/E7. BNIP3L protein was observed in 46.7% (14/30) lung cancer tissues, while 100% (12/12) in normal lung tissues. The difference was significant in statistics (P< 0.05). (2) BNIP3L mRNA was detected in 4 lung cancer cell strains; and there existed no obvious discrepancy of the amount between these cell strains and HBE4-E6/E7. Absence or decrease of BNIP3L mRNA was observed in 26.7%(8/30) of lung cancer tissues. The average quantity of BNIP3L mRNA was 0.404+/-0.070 in lung cancer tissues, while 0.575+/-0.065 in paired normal lung tissues. The difference was significant in statistics (P< 0.05). In all the cancerous cell strains and tissues with BNIP3L mRNA, the products of RT-PCR were as long as those from their control samples in size, including the entire coding region, and no variation of BNIP3L gene structure such as absence, rearrangement, aberrant splicing were detected.(3) No point mutation was detected in all 6 exons of BNIP3L gene in 4 lung cancer cell strains and 30 tissues. CONCLUSION: BNIP3L protein expression was down-regulated in lung cancer, which might be involved in the occurrence and/or development of lung cancer. The down-regulation of BNIP3L protein expression in lung cancer was partly caused by the down-regulation of its transcription. The variation of gene structure may be not the reason of BNIP3L inactivity in lung cancer.