OBJECTIVETo explore the mechanism of action of RbAp46 gene on leukemic cells.

METHODSK562 leukemic cells and SHG44 glioma cells were transfected with eukaryotic expression vector carrying full-length cDNA of RbAp46 driven by the cytomegalovirus promoter mediated by lipofectamine transfection reagent. Empty vector were transfected at the same time as control. G418-resistant colonies were selected after 3 weeks culturing. Series genes were amplified using RT-PCR. Growth curve and colony formation assays were performed.

RESULTSThe number of K562/RbAp46 and K562/CMV cells were (90.00 +/- 8.40) x 10(4) and (119.58 +/- 9.87) x 10(4), respectively after 4 days growth, and SHG44/RbAp46 and SHG44/CMV cells were (89.13 +/- 4.88) x 10(4) and (149.42 +/- 10.83) x 10(4), respectively after 5 days growth. Seven-day yields of K562/RbAp46 and K562/CMV cell colonies were 131.67 +/- 15.57 and 250.33 +/- 26.31, respectively (P < 0.01), while those of SHG44/RbAp46 and SHG44/CMV cells were 50.78 +/- 6.77 and 206.67 +/- 37.18, respectively (P < 0.01). The fraction of K562/RbAp46 and K562/CMV cells in S phase was (48.88 +/- 4.35)% and (62.78 +/- 4.78)% (P < 0.01), and in G(0)/G(1) phase was (29.10 +/- 4.14)% and (22.40 +/- 2.43)%, respectively (P < 0.05), and that of SHG44/RbAp46 and SHG44/CMV cells in G(0)/G(1) phase was 65.6% and 48.8%, and in S phase was 22.6% and 38.4%, respectively. Insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1) gene was induced to express only in the RbAp46-over expressing K562 cells and was not in SHG44 cells.

CONCLUSIONA regulatory pathway between RbAp46 and IGFBP-rP1 genes might exit in K562 leukemic cells.

Carrier Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; K562 Cells ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transfection

To establish leukemic cell lines stably transfected by RbAp46 gene, electroporation was performed after optimizing the transfection condition for suspended cells. Under conditions of low voltage and high capacitance, RbAp46 was transfected into U937 by electroporation. Individual clones selected with G418 for 3 weeks were isolated. The integration and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis, respectively. The subclone expressing high level of RbAp46 was then established. Viability of transfected cells was assayed by trypan blue exclusion. Cell number was counted daily to determine the growth rate. The results showed that growth rate of U937 cell lines expressing exogenous RbAp46 was about 50% lower than that in control. It is concluded that leukemic cell lines stably expressing exogenous RbAp46 were established and overexpression of RbAp46 inhibits the growth of U937 leukemic cells.
Blotting, Western ; Carrier Proteins ; genetics ; Cell Proliferation ; Electroporation ; Humans ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transfection ; U937 Cells ; WT1 Proteins ; genetics

OBJECTIVEE2F-1 and Rb are involved in cell cycle regulation. This study was to illustrate the mechanism of transformation from benign papillomatosis to ductal carcinoma in situ (DCIS) of the breast in relation to E2F-1 and Rb expression.

METHODSIn situ hybridization (ISH) was used to determinate the expression of E2F-1 and Rb mRNA of mild papillomatosis (MP, n = 40), severe papillomatosis (SP, n = 40) and DCIS (n = 40). Immunohistochemistry (IHC) was used to examine the expression of E2F-1 and Rb protein.

RESULTSThe positive rate of E2F-1 mRNA expression in MP, SP and DCIS was 17.5%, 45.0% and 80.0%, and that of E2F-1 protein expression was 20.0%, 47.5% and 77.5%, respectively. There were significant differences among the three groups (P < 0.01), and between any two groups (P < 0.01). The positive rate of Rb mRNA expression in MP, SP and DCIS was 90.0%, 50.5% and 20.0%, and that of Rb protein expression was 85.0%, 52.5% and 22.5%, respectively, with statistically significant difference similar with that of E2F-1. With the progression of papillomatosis to DCIS, the expression of E2F-1 mRNA and protein increased, while that of Rb decreased. The protein expression by IHC was positively correlated with the mRNA expression by ISH. However, that of E2F-1 was negatively correlated with Rb.

CONCLUSIONE2F-1 and Rb might provide a valuable basis for screening high risk papillomatosis and new target of gene therapy for pre-cancerous lesions of the breast.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; Cell Cycle Proteins ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; E2F Transcription Factors ; E2F1 Transcription Factor ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Retinoblastoma ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Papilloma ; genetics ; metabolism ; Precancerous Conditions ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Retinoblastoma Protein ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics

WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.
Animals ; Carrier Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; DNA-Binding Proteins ; metabolism ; Erythroid-Specific DNA-Binding Factors ; GATA1 Transcription Factor ; Gene Expression Regulation ; Humans ; Leukemia ; etiology ; genetics ; NF-kappa B ; metabolism ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transcription Factors ; metabolism ; Transcription, Genetic ; WT1 Proteins ; physiology

To explore the feasibility of real-time quantitative PCR (QRT-PCR) for selecting cell strains which overexpress a certain transgene, expression level of RbAp46 was detected in transfected cell strains by using optimal real-time PCR with SYBR Green I. Meanwhile, semi-quantitative RT-PCR and Western blot were performed to compare with the QRT-PCR. The results showed that values of RbAp46(N) were 2064.42 +/- 253.47, 860.94 +/- 291.07, 234.456 +/- 31.08, 18.17 +/- 5.14 and 1.46 +/- 0.54 in K562/RbAp46, K562/CMV, SHG44/RbAp46 monoclone, SHG44/RbAp46 multiclone and SHG44/CMV, respectively. The results were consistent with that determined by semi-quantitative RT-PCR and Western blot. It is concluded that QRT-PCR provides a highly efficient and reproducible method for selection of transfected cell subclones at different level of transgene expression.
Blotting, Western ; Carrier Proteins ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; Nuclear Proteins ; genetics ; metabolism ; Organic Chemicals ; chemistry ; RNA, Neoplasm ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transfection ; Transgenes ; genetics

OBJECTIVETo investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients.

METHODSReal-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders.

RESULTSThe M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression.

CONCLUSIONRbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.

Adolescent ; Adult ; Aged ; Carrier Proteins ; genetics ; metabolism ; Child ; Female ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the expression of retinoblastoma-associated protein 46 (RbAp46) or RbAp 46 mRNA in bone marrow mononuclear cells (BMMNC) of acute leukemia (AL) patients and determine whether the expression is related to the classification and prognosis of ALs.

METHODSThe expression of RbAp46 protein in BMMNC was detected by Western blot in 46 AL patients and the expression of RbAp46 mRNA in BMMNC by semi-quantitative RT-PCR in 22 AL patients. The indirect immunofluorescence staining technique was applied to the localization of RbAp46 protein in BMMNC both in leukemia patients and control subjects.

RESULTS(1) Both RbAp46 protein and mRNA were expressed in AL BMMNC and no significant difference was found among different leukemia types. (2) The expression of RbAp46 protein was lower in AL patients with high-degree tumor burden than in those with low-degree tumor burden (mean A, 93.4 +/- 37.2 vs 127.2 +/- 15.8, P < 0. 05). (3) The expression of RbAp46 protein was lower in refractory leukemia than those in non-refractory leukemia (mean A, 87.1 +/- 33.8 vs 126.6 +/- 21.2, P < 0. 05). (4) The expression of RbAp46 mRNA was lower in AL patients with high-degree tumor burden than in those with low-degree tumor burden (mean A R, 0.19 +/- 0.08 vs 0.31 +/- 0.12, P < 0. 05). (5) RbAp46 protein was mainly localized in nucleus of BMMNC in both AL patients and control subjects.

CONCLUSIONBoth RbAp46 protein and mRNA are expressed in AL patients BMMNC. The downregulation of RbAp46 expression is associated with high leukemic burden and refractory to treatment. RbAp46 gene might be a tumor suppressor gene for leukemia.

Acute Disease ; Adolescent ; Adult ; Aged ; Blotting, Western ; Bone Marrow Cells ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Leukemia ; blood ; pathology ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; metabolism ; Prognosis ; RNA, Messenger ; genetics ; metabolism ; Retinoblastoma Protein ; genetics ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

No abstract available.
Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 22 ; Chromosomes, Human, Pair 9 ; Female ; Fusion Proteins, bcr-abl/genetics ; Humans ; In Situ Hybridization, Fluorescence ; Karyotype ; Middle Aged ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/*genetics/pathology ; Retinoblastoma Binding Proteins/genetics ; *Translocation, Genetic ; Ubiquitin-Protein Ligases/genetics

Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16(INK4A) and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16(INK4A) and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16(INK4A), but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16(INK4A)-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16(INK4A) and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating PML stability.
Cell Line, Tumor ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; antagonists & inhibitors ; genetics ; physiology ; DNA-Binding Proteins ; deficiency ; genetics ; physiology ; Down-Regulation ; Fibroblasts ; Gene Expression Regulation ; Humans ; Intranuclear Inclusion Bodies ; metabolism ; MAP Kinase Signaling System ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Promyelocytic Leukemia Protein ; Protein Isoforms ; Protein Stability ; RNA Interference ; Retinoblastoma Protein ; antagonists & inhibitors ; genetics ; physiology ; Transcription Factors ; deficiency ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Protein p53 ; physiology ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; Ubiquitination ; ras Proteins ; metabolism

To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma.
3' Untranslated Regions ; ATP Binding Cassette Transporter, Sub-Family G, Member 2/antagonists & inhibitors/genetics/*metabolism ; Antagomirs/metabolism ; Antineoplastic Agents/toxicity ; Apoptosis/drug effects ; Base Sequence ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/drug effects ; Gene Silencing ; Humans ; MicroRNAs/antagonists & inhibitors/genetics/*metabolism ; Neoplasm Proteins/antagonists & inhibitors/genetics/*metabolism ; Neoplastic Stem Cells/*metabolism ; Retinoblastoma/metabolism/pathology ; Sequence Alignment ; Transfection