A differential gene expression profiles by cDNA microarrays in endometrioid endometrial carcinoma: a preliminary study / 부인종양

OBJECTIVE: Endometrial carcinoma is the most common gynecological malignant disease in industrialized countries. However, the molecular bases for endometrial tumoriogenesis are not clearly elucidated. Our hypothesis is that there may be some difference in gene expression patterns between normal endometrium and endometrial cancer lesion. In this study, we analyzed the difference of gene expression profile with cDNA microarray. METHODS: Normal endometrial tissues and cancer lesions were gathered from three patient with endometrioid endometrial cancer. cDNA microarray technique (KNU 4.8K chip) was applied to screen the different gene expression profiles. RESULTS: Many genes such as interleukin-1 receptor-associated kinase 1 (IRAK1), bifunctional apoptosis regulator (BFAR), paraneoplastic antigen MA2 (PNMA2), zinc finger protein 257 (ZNF257), ras homolog gene family, member F (in filopodia) (ARHF), cell division cycle 27 (CDC27) were over-expressed in the endometrial cancer tissue. The genes were down-regulated in the endometrial cancer samples included fibronectin 1 (FN1), meiotic checkpoint regulator (MCPR), transforming growth factor beta-stimulated protein TSC-22 (TSC22), programmed cell death 4 (neoplastic transformation inhibitor) (PDCD4), transcript variant 2, matrix metalloproteinase 2 (MMP2), insulin-like growth factor binding protein 4 (IGFBP4), retinoblastoma binding protein 7 (RBBP7), insulin-like growth factor binding protein 3 (IGFBP3), downregulated in ovarian cancer 1 (DOC1). CONCLUSION: The result of this analysis supports the hypothesis that the endometrial cancer tissue has distinct gene expression profile from normal endometium. But, the vaildation of gene expression with RT-PCR and the further study are needed.

RbAp46 gene activates the expression of IGFBP-rP1 gene in K562 leukemic cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of action of RbAp46 gene on leukemic cells.</p><p><b>METHODS</b>K562 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>A regulatory pathway between RbAp46 and IGFBP-rP1 genes might exit in K562 leukemic cells.</p>

Application of real-time quantitative PCR in selection of transfected cell strains for transgenic overexpression / 中国实验血液学杂志

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.

Expressive profile of retinoblastoma-associated protein 46 and its clinical significance in acute leukemias / 中华血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>Both 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.</p>

Detection of RbAp46 expression in bone marrow cells of leukemia patients by real-time quantitative RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients.</p><p><b>METHODS</b>Real-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>RbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.</p>

Establishment and characterization of leukemic cell line U937 stably expressing exogenous RbAp46 / 中国实验血液学杂志

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.

WT1-mediated pathway of transcriptional regulation and leukemia / 中国实验血液学杂志

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.