Quantitative analysis of gene expression for vascular endothelial growth factor and its application / 中国实验血液学杂志

Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template (T-VEGFDelta) was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample. The surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 x 10(4) to 2 x 10(5) molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 x 10(5), 12.6 x 10(5), 3.6 x 10(5), and less than 1.0 x 10(5)/microg total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.

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.

Report of a case of congenital plasminogen activator inhibitor-1 deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To report a patient with congenital plasminogen activator inhibitor-1 (PAI-1) deficiency and explore its molecular mechanism.</p><p><b>METHODS</b>The activities of tissue plasminogen activator (tPA), alpha(2) antiplasmin (alpha(2)AP) and PAI-1 were measured by the methods of chromogenic substrate, the antigens of tPA and PAI-1 were measured by ELISA. PAI-1 gene was studied by PCR product sequencing and restriction endonuclease ana-lysing.</p><p><b>RESULTS</b>In the present patient, the euglobulin clot lysis time was 70 minutes and was corrected to normal range after added 50 ng/ml PAI-1 to his plasma. The activities of t-PA, alpha(2)AP, and factor were normal; the activity and antigen of PAI-1 in plasma were both significantly decreased. Nucleotide sequence analysis revealed that the patient had a heterozygous missense mutation in exon 2, a G to A transition at nucleotide 43. The possibility of gene polymorphism was excluded by restriction endonuclease analysing.</p><p><b>CONCLUSIONS</b>It is the first patient with congenital PAI-1 deficiency reported in China. The PAI-1 deficiency in the patient may be caused by compound heterozygosity, one of which is the G to A transition at nt43, a new mutation in congenital PAI-1 deficiency.</p>

The expression of CXCR4 on acute leukemia cells and its implication for extramedullary infiltration / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of CXCR4 in acute leukemic cells and its clinical significance.</p><p><b>METHOD</b>Bone marrow samples from 73 acute leukemia patients and leukemic cell lines were investigated by flow cytometry (FCM), the expression of SDF-1 in human marrow stromal cells and meninges were studied by using reverse transcription polymerase chain reaction (RT-PCR). Adhesion, migration and invasion of U937, NB4 and K562 cells were studied in vitro.</p><p><b>RESULTS</b>The expression rates of CXCR4 in ALL and AML patients was 65.6% and 17.1%, respectively. And it was 0.2%, 41.0% and 52.0% in K562, U937 and NB4 cells, respectively. The extramedullary infiltration rates were 61.9% and 18.2% for CXCR4 positive and negative groups of ALL, respectively (P < 0.05); while in AML, the number of peripheral white blood cells in CXCR4 positive group was lower than that in CXCR4 negative group (P < 0.05). SDF-1alpha could enhance the adhesion, migration and invasion capacity of leukemic cells in vitro.</p><p><b>CONCLUSION</b>Overexpression of CXCR4 in AL cells might be the molecular mechanism of extramedullary infiltration in leukemia.</p>

Effects of tributyrin on SHI-1 leukemia cells in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of tributyrin (TB), a histone deacetylase inhibitor, on the growth, differentiation and apoptosis of SHI-1 leukemia cells and explore its possible mechanism.</p><p><b>METHOD</b>Cell proliferation and viability were determined by cell counting, trypan blue dye exclusion. Cell morphological analysis, Annexin binding, DNA electrophoresis, expression of CD11b and CD14, NBT reduction were performed to evaluate differentiation and apoptosis of SHI-1 cells. The level of acetylated histone H3 was detected by Western blot and p21(WAF1/CIP1) expression by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>TB inhibited the proliferation and viability of SHI-1 cells in a time-dose dependent manner. The morphology of SHI-1 cells cultured in the presence of 0.1 mmol/L TB for 72 hs was more mature with higher NBT positivity and up-regulated expressions of CD11b and CD14 than that of control group. Exposed to 0.5 - 1.0 mmol/L TB for 48 hs, SHI-1 cells exhibited the morphological hallmarks of apoptosis, the increasing of Annexin binding and the DNA ladder on gel electrophoresis. The level of acetylated histone H3 and p21(WAF1) mRNA extracted from SHI-1 cells were increased by the treatment of TB.</p><p><b>CONCLUSION</b>TB can inhibit proliferation, induce differentiation and apoptosis of SHI-1 cells. The mechanism may associate with its up-regulation of acetylated histone and the expression of p21(WAF1).</p>

Molecular biological study of glycoprotein IX gene defect in Bernard-Soulier syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis.</p><p><b>METHODS</b>Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting.</p><p><b>RESULTS</b>The patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma.</p><p><b>CONCLUSION</b>The mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.</p>

Establishment of urokinase receptor gene antisense RNA transfer system and its application in leukemia research / 中国实验血液学杂志

Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.

Aldehyde-dehydrogenase gene-transduced hematopoietic cell line K562 overcomes the cytoxicity of cyclophosphamide in vitro / 中国实验血液学杂志

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cells lines in vitro, this study tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. Results showed that a retroviral vector was used to transduce full-length human ALDH1 cDNA into human hematopoietic cell line K562 that was then tested for resistance to 4-hydroxycyclophosphamide (4-HC), an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced K562 cells (50% inhibition concentration, IC50 = 10 micro mol/L). These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

Change of WT1 Gene Expression during Induction of Differentiation of NB4 Cell Line / 中国实验血液学杂志

In order to study the relation between WT1 gene expression and differentiation of NB4 leukemic cells, a competitive RT-PCR method was established by using recombinant DNA technique to detect the expression of WT1 gene quantitatively during differentiation of NB4 leukemic cells induced by retinoic acid. The expression of CD11b was simultaneously determined by an indirect immunofluorescence staining technique and flow cytometry. Our results showed that WT1 gene expression rapidly decreased during the differentiation of NB4 cells. The molecular number of WT1 gene in 1 micro g total RNA was 4 x 10(6), 1.56 x 10(6) and 0.4 x 10(6), respectively, prior to and at 12 or 24 hours after exposure to retinoic acid, and it was in accordance with the change of CD11b. These data suggest that the abnormally high expression of WT1 gene in leukemic cells was associated with the block of cell differentiation. The detection of WT1 expression with competitive RT-PCR combined with measure of CD antigens will contribute to study on the differentiation induction of leukemic cells.