Dlx5, the mouse homologue of the human-imprinted DLX5 gene,is biallelically expressed in the mouse brain

The mouse Dlx5 gene encodes a distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of mouse embryo and is located on chromosome 6, which is the syntenic region to the human chromosome 7q21–q31 imprinting cluster. Recently, its human homologue, DLX5, was identified to be imprinted and maternally expressed, at least in normal human lymphoblasts and in brain tissues. In our study, we analyzed the imprinting status of mouse Dlx5 by RT-PCR, first in the F1 of a reciprocal cross between two different mouse strains, and second in heterozygous Dlx5 mutant mice. Both approaches revealed that mouse Dlx5 followed a biallelic pattern of expression in brain tissue and in testis. Our findings suggest that the Dlx5 gene escapes genomic imprinting, at least in mice of certain genetic backgrounds.     

Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction

Idiopathic fetal growth restriction (FGR) is often associated with placental insufficiency. Previously, we isolated and characterized homeobox gene DLX4 from the placenta and provided evidence that DLX4 may regulate placental development. Here, we have investigated whether DLX4 expression levels were altered in idiopathic FGR. FGR-affected placentae were collected based on strict clinical criteria. DLX4 mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Initial RT-PCR results showed a qualitative increase in DLX4 mRNA in both FGR-affected placentae and gestation-matched controls. Real-time PCR showed a 3-fold increase in DLX4 mRNA levels in FGR-affected placentae compared with gestation-matched controls (P < 0.005). Western immunoblotting using a rabbit DLX4 polyclonal antibody revealed significantly increased levels of DLX4 protein in term FGR-affected placentae compared with term controls [5500.1 +/- 21.8 (n = 10) versus 3533.2 +/- 22.4 (n = 10); P < 0.001]. Qualitative immunohistochemical analyses of term placentae showed moderately increased immunoreactivity for DLX4 antigen in the FGR-affected placentae in syncytiotrophoblasts, residual cytotrophoblast cells and endothelial cells of the fetal capillaries compared with gestation-matched control term placentae. We conclude that the increased expression of homeobox gene DLX4 may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.     

Identification of a mutation in DLX3 associated with tricho-dento- osseous (TDO) syndrome

Tricho-dento-osseous syndrome (TDO) is an autosomal dominant disorder characterized by abnormal hair, teeth and bone. The main clinical manifestations of TDO include taurodontism, enamel hypoplasia, kinky, curly hair at birth and increased thickness and density of the cranial bones. These pleiotropic clinical features suggest the role of a developmental gene modulating epithelial-mesenchymal interactions. We recently mapped the TDO locus to chromosome 17q21, a region that includes two members of the distal-less homeobox gene family, DLX3 and DLX7. In this paper we describe genomic cloning and sequencing of both human DLX3 and DLX7 and identification of a 4 bp deletion in human DLX3 which correlates with the TDO phenotype in six families. The observed mutation is predicted to cause a frameshift and premature termination codon, resulting in a functionally altered DLX3. This first report of a human mutation in the DLX genes is consistent with murine studies indicating their important role in the development of hair, teeth and bone.      

A homeobox gene related to Drosophila distal-less promotes ovarian tumorigenicity by inducing expression of vascular endothelial growth factor and fibroblast growth factor-2

Homeobox genes control developmental patterning and are increasingly being found to be deregulated in tumors. The DLX4 homeobox gene maps to the 17q21.3-q22 region that is amplified in some epithelial ovarian cancers. Because amplification of this region correlates with poor prognosis, we investigated whether DLX4 overexpression contributes to aggressive behavior of this disease. DLX4 was not detected in normal ovary and cystadenomas, whereas its expression in ovarian carcinomas was strongly associated with high tumor grade and advanced disease stage. Overexpression of DLX4 in ovarian cancer cells promoted growth in low serum and colony formation. Imaging of mice bearing intraperitoneal tumors revealed that DLX4 overexpression substantially increased tumor burden. Tumors that overexpressed DLX4 were more vascularized than vector-control tumors. Conditioned medium of DLX4-overexpressing tumor cells was more effective than medium conditioned by vector-control cells in stimulating endothelial cell growth. These observations were associated with the ability of DLX4 to induce expression of vascular endothelial growth factor as well as intracellular and secreted isoforms of fibroblast growth factor-2. Moreover, increased levels of these fibroblast growth factor-2 isoforms induced vascular endothelial growth factor expression in tumor cells. This study reveals a novel role for a homeobox gene in ovarian tumorigenicity by its induction of a proangiogenic, growth-stimulatory molecular program.     

Homeodomain protein DLX4 facilitates nasopharyngeal carcinoma progression via up‐regulation of YB‐1

Nasopharyngeal carcinoma (NPC) is a malignant tumor in nasopharynx tissues and lacks effective treatment strategies. Dysregulation of distal‐less homeobox 4 (DLX4) participates in the development of tumors. Understanding the regulatory mechanism of DLX4 in NPC progression may address this issue. Here, we first identified an up‐regulation of DLX4 in NPC cell lines compared to normal epithelial cells. Data from colony formation and transwell assays showed that knockdown of DLX4 inhibited cell proliferation and invasion of NPC, respectively. Moreover, DLX4 knockdown blocked the cell cycle of NPC at G1 phase, suggesting the antitumor effect of DLX4 knockdown on NPC. The downstream target of DLX4 was identified as Y‐box binding protein 1 (YB‐1), whose expression was increased by over‐expression of DLX4, while decreased by knockdown of DLX4. The binding capacity between DLX4 and YB‐1 was verified by chromatin immunoprecipitation (ChIP), and the result showed that DLX4 could not directly bind to the promoter of YB‐1. Mechanically, YB‐1 over‐expression reversed the effects of DLX4 knockdown on cell proliferation, cell cycle arrest and cell invasion of NPC. In conclusion, our findings indicated that DLX4 promoted NPC progression via up‐regulation of YB‐1, which would shed light on therapeutic schedule in NPC.     

不同处理因素对儿童白血病细胞抗凋亡基因表达的影响

目的研究儿童白血病骨髓基质细胞(BMSCs)、VLA-4抗体、化疗药物VP-16、VLA-4抗体联合VP-16对各组白血病细胞抗凋亡基因表达的影响并探讨相关机制。方法收集初治儿童急性白血病骨髓标本,分离单个核细胞,采用RT-PCR方法检测各组白血病细胞抗凋亡基因Survivin、Bcl-2的表达。结果29例儿童急性白血病中,23例有Sur-vivin基因的表达,表达率高达79%;28例有Bcl-2基因的表达。BMSCs组抗凋亡基因Survivin、Bcl-2表达量最多,骨髓基质上清组仅次于BMSCs组,上述两组之间差异无统计学意义,但与白血病细胞单独培养组比,均有显著差异。抗体组、药物组、抗体加药物组Survivin、Bcl-2基因的表达均显著降低,差异均有统计学意义。抗体加药物组的表达量最少,与BMSCs组比,Survivin表达降低了56%左右,而Bcl-2表达量降低了71%。结论VLA-4抗体和VP-16可下调白血病细胞抗凋亡基因Survivin、Bcl-2的表达,两者联合应用,下调幅度更大。     

DLX1联合BMP9促进人骨肉瘤细胞向成骨分化

目的探讨DLX1联合BMP9可否影响人骨肉瘤细胞MG63的成骨分化。方法用构建有DLX1和BMP9基因的重组腺病毒Ad DLX1和Ad BMP9,单独或联合感染MG63细胞,分为DLX1组(Ad DLX1+AdRFP感染组)、DLX1+BMP9组(Ad DLX1+Ad BMP9感染组)、BMP9组(Ad BMP9+AdRFP感染组)、RFP组(AdRFP感染组)。用RT-PCR和Western blot验证DLX1和BMP9的表达情况,Transwell实验检测细胞迁移、侵袭能力;碱性磷酸酶(ALP)染色和读数分析细胞早期成骨能力,茜素红染色检测细胞晚期成骨能力,Western blot检测骨桥蛋白(OPN)蛋白表达水平。结果 Ad DLX1和Ad BMP9感染MG63细胞后,DLX1和BMP9的表达水平上调(P0.05);与RFP组相比,DLX1组、DLX1+BMP9组和BMP9组细胞迁移能力和侵袭能力下降(P0.05),DLX1+BMP9组细胞迁移能力和侵袭能力下降更为明显(P0.05);ALP活性、钙盐沉积和OPN蛋白表达在DLX1+BMP9组和BMP9组增加(P0.05),且DLX1+BMP9组较BMP9组显著增强(P0.05)。结论 DLX1与BMP9联合作用可抑制骨肉瘤细胞迁移和侵袭,同时可诱导骨肉瘤细胞向成骨分化。     

Hu-ets-1 gene in congenital leukemia with t(11;19)(q23;p13)

Cytogenetic analysis of the leukemic cells from a 1-day-old baby with an acute myelomonocytic leukemia revealed them to contain a chromosome change of t(11;19)(q23;p13). Molecular studies using a 980 bp HindIII/HpaI digested v-ets probe showed no DNA rearrangements, deletions, or amplification in the leukemic cells, including the JH immunoglobulin and T-cell receptor ( or β) genes. The findings indicate that the leukemic cells with t(11;19)(q23;p13) appear not to contain a transposition or rearrangement of the protooncogene Hu-ets-1 located at 11q23, as previously described in leukemic cells with t(4;11)(q21;q23) and t(9;11)(p22;q23). The leukemic cases with t(11;19)(q23;p13) studied by us showed a phenotype compatible with their myelomonocytic nature, although it is possible that other cases may have a lymphoid phenotype.     

远端同源异型盒2和叉头盒O3a在大鼠脑皮质发育过程中的分布与表达

目的 了目的 探讨远端同源异型盒2（Dlx2）和叉头盒O3a（FoxO3a）在SD大鼠脑皮质发育过程中的表达特点。 方法 应用Real-time PCR法检测Dlx2 mRNA和FoxO3a mRNA分别在受孕11d（E11）、E13、E15、E17、E19及生后1d（P1）的表达情况;应用免疫组织化学技术显示Dlx2和FoxO3a在E11、E13、E15、E17、E19及P1蛋白的表达情况。 结果 FoxO3a与Dlx2 mRNA在E11～P1均有表达,而FoxO3a mRNA表达高峰明显早于Dlx2 mRNA;Dlx2 mRNA表达在E15～E17时大幅度上升,而在此之后始终保持这一高水平的表达;Dlx2 mRNA与FoxO3a mRNA的表达在E15～P1呈现出一致的变化趋势;Dlx2基因在E19时出现mRNA水平的高表达而未见蛋白表达。 结论 Dlx2和FoxO3a基因在胚胎后期鼠脑皮质中均有表达,且其表达趋势具有一定的一致性;两基因的分布随皮质分层而改变。     

Molecular and phenotypic analysis of Philadelphia chromosome-positive bilineage leukemia: possibility of a lineage switch from T-lymphoid leukemic progenitor to myeloid cells

The occurrence of acute bilineage leukemia is thought to be the malignant transformation of a myeloid or lymphoid leukemic progenitor with the potential to differentiate into the other lineages; however, the mechanisms of this lineage switch are not well understood. Here, we report on the extremely rare case of adult Philadelphia chromosome-positive acute bilineage leukemia, which is characterized by T-cell acute lymphoblastic leukemia and acute myelomonocytic leukemia. Chromosome analysis showed 46,XY,del(7)(p11.2),t(9;22)(q34;q11.2) in all metaphases and a minor BCR/ABL chimeric gene was detected in these leukemic cells by PT-PCR. When the CD5+ and CD5- cells were sorted, a fusion gene of BCR/ABL and the same clonally rearranged band of a T-cell receptor (TCR) gene were detected in both populations. Nucleotide sequencing of the TCR-gamma gene revealed the clonal rearrangement of the V8-JGT2 complex in both populations. Overexpression of PU.1, which plays a fundamental role in myelomonocyte development, was found in the sorted CD34+CD7+ and CD5-, but not CD5+ cells. These results suggest that leukemic progenitor cells in the T-lineage with the del(7) and t(9;22) have the potential to differentiate into myeloid lineage, and that enforced PU.1 expression may contribute in part of this phenomenon.     

A common DLX3 gene mutation is responsible for tricho-dento-osseous syndrome in Virginia and North Carolina families.

Tricho-dento-osseous syndrome (TDO) is characterised by a variable clinical phenotype primarily affecting the hair, teeth, and bone. Different clinical features are observed between and within TDO families. It is not known whether the variable clinical features are the result of genetic heterogeneity or clinical variability. A gene for TDO was localised recently to chromosome 17q21 in four North Carolina families, and a 4 bp deletion in the human distal-less 3 gene (DLX3) was identified in all affected members. A previous genetic linkage study in a large Virginia kindred with TDO indicated possible linkage to the ABO, Gc, and Kell blood group loci. To examine whether TDO exhibits genetic heterogeneity, we have performed molecular genetic analysis to determine whether affected members of this Virginia kindred have the DLX3 gene deletion identified in North Carolina families. Results show that affected subjects (n=3) from the Virginia family have the same four nucleotide deletion previously identified in the North Carolina families. A common haplotype for three genetic markers surrounding the DLX3 gene was identified in all affected subjects in the North Carolina and Virginia families. These findings suggest that all people with TDO who have been evaluated have inherited the same DLX3 gene deletion mutation from a common ancestor. The variable clinical phenotype observed in these North Carolina and Virginia families, which share a common gene mutation, suggests that clinical variability is not the result of genetic heterogeneity at the major locus, but may reflect genetic heterogeneity at other epigenetic loci or contributing environmental factors or both.     

Split hand/foot malformation due to chromosome 7q aberrations(SHFM1): additional support for functional haploinsufficiency as the causative mechanism

We report on three patients with split hand/foot malformation type 1 (SHFM1). We detected a deletion in two patients and an inversion in the third, all involving chromosome 7q21q22. We performed conventional chromosomal analysis, array comparative genomic hybridization and fluorescence in situ hybridization. Both deletions included the known genes associated with SHFM1 (DLX5, DLX6 and DSS1), whereas in the third patient one of the inversion break points was located just centromeric to these genes. These observations confirm that haploinsufficiency due to either a simultaneous deletion of these genes or combined downregulation of gene expression due to a disruption in the region between these genes and a control element could be the cause of the syndrome. We review previously reported studies that support this hypothetical mechanism.