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8-Chloro-adenosine (8-CI-Ado) is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt) and H1299 (p53-depleted) to 8-CI-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-CI-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

To investigate the relationship of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus(ICTEV), 84 samples from patients with ICTEV were used in the study. Mutation in the coding region of HOXD13 was detected by denaturing gradinent electrophoresis. The mRNA and protein levels of HOXD13 and FHL1 were evaluated by RT-PCR and immunohistochemistry, respectively. The binding site of FHL1 to HOXD13 predicted by PMATCH software was validated by EMSA( Electrophoretic mobility shift assay,EMSA).No mutation was found in the coding region of HOXD13 in 84 samples from patients with ICTEV. Both HOXD13(33.3%) and FHL1(46.6%) were down-regulated in ICTEV muscle tissue. The result of EMSA showed that the special binding band appeared when HOXD13 existed. The results shows that HOXD13 gene mutation was not involved in outbreak in idiopathic congenital talipes equinovarus, but changes of HOXD13 and FHL1 gene expression related to the development of talipes equinovarus malformation. HOXD13 might play an role in ICTEV through regulating FHL1 expression.

Retinoic acid can induce teratogenesis of the fetus of many animals including human, and its biological activities are induced by a serious of different retinoic acid accepters and their ligands. The retinoic acid acceptor RAR plays key roles in the teratogenesis, and the ligands of RAR are strong teratogens. The intensity sequence of the relative teratogenesis is ligandalpha, ligandbeta and ligandgamma. The ligands of the retinoic acid acceptor RXR cannot induce teratogenesis, but they can enhance the teratogenesis of the RAR stimulus. The retinoic acid acceptors can also affect the development of the fetus by adjusting the expression of the other genes. The relations between the gene mutation of the retinoic acid acceptor, various retinoic acid acceptors and their ligands and teratogenesis of retinoic acid are summarized in this article. In addition, the regulations of the retinoic acid acceptors to the other genes are also discussed.

Loss of tumor suppressor genes and apoptosis-associated genes was common event in laryngeal squamous cell carcinoma (LSCC). Apaf-1 (apoptotic protease activating factor-1) is a key factor in cytochrome C-dependent apoptotic pathway. To investigate the effect of Apaf-1 in progression of LSCC, we analyzed Apaf-1 from DNA and RNA levels. Semi-quantitative RT-PCR analysis of Apaf-1 mRNA showed that over 40 percent of LSCCs (11/27) were low expression compared to the para-neoplastic laryngeal tissues (PNTs). The results of CGH indicated loss in 2 of 18 cases but no amplification on chromosome 12q22-23. The LOH frequencies of D12S327, D12S1657, D12S393, D12S1706, D12S346 on 12q22-23 were 18.2%, 13.6%, 18.2%, 22.2% and 16.6%, respectively in 72 matched samples of LSCCs and PNTs. Methylation-specific PCR displayed that all of 11 LSCCs, whose expression of Apaf-1 mRNA down-regulated, were methylated in promoter regions. In contrast, only 1 of 16 LSCCs with no changes in Apaf-1 mRNA levels was methylated (chi2 test, P=0.0001). The results implied that abnormal expression of Apaf-1 participates in the genesis of LSCCs and decreased expression of Apaf-1 mRNA were not mainly due to the deletion of Apaf-1 gene. The Apaf-1 DNA methylation in promoter region might contribute to the decreased transcription of Apaf-1 in LSCCs.

To assess the relationship of deletion of p15 and p16 gene and EGFR gene amplification in laryngeal squamous cell carcinoma (LSCC). DNA was extracted from fresh tumor. Deletion of p15 exon 2(p15E2) and p16 exon 2(p16E2) in 30 cases of LSCC was detected by polymerase chain reaction (PCR) technique. Amplification of EGFR gene in 30 cases of LSCC was detected by FISH. The rate of p15E2 deletion in 30 cases was 13.3(4/30), and that of p16E2 was 16.7% (5/30). p15E2 and p16E2 codeletion rate was 6.7% (2/30). The rate of EGFR gene amplification in 30 cases was 30% (9/30), and was amplified 2 to 8 fold. Homozygous deletion of p16E2 and p15E2 and codeletion is related with amplification of EGFR gene (P = 0.000018), and may play an important role to oncogenesis and malignant progression in LSCC.

To observe expression of Wilms' tumor-1 (WT1) gene in the different subtypes of myelodysplastic syndrome (MDS), and to explore the regularity of expression of WT1 gene in the process of MDS transforming into acute leukemia (AL).The method of reverse transcriptase-polymerase chain reaction (RT-PCR) was used, the levels of WT1 gene's presentation in different types of montagers of MDS were analyzed, and the relationship between the level and the clinical characteristic was analyzed. At the same time, the expression of WT1 gene in AL patients, post-MDS-AL patients and normal controls were examined.The positive rate of WT1 gene expression in 49 patients with MDS was 22.4 percent (11/49), refractory anemia (RA) was 0(0/13), RA with excess of blast (RAEB) was 25.0 percent (6/24); RAEB in transformation (RAEB-t) was 41.7 percent (5/12). The positive rates of WT1 expression were gradually increased in three types of MDS (P0.05 and P0.01). The positive rates of WT1 expression were higher in AL and post-MDS-AL patients (48.5 percent, 32/66 and 50.0 percent, 4/8) than that in MDS patients (P0.01). There was no expression of WT1 gene in normal control.There is a relatively high expression rate of WT1 gene in RAEB, RAEB-t of MDS, but relatively low expression rate in RA. The method of RT-PCR, is high sensitiveness and specificity, can trustful be used in the progression of monitoring MDS' progression and its transformation into AL.

To investigate the association of p15 and p16 genes deletion, and STK15 gene overexpression with laryngeal squamous cell carcinoma (LSCC).The cancer tissue and surrounding normal tissue were taken during operation from 50 cases of LSCC who had undergone neither chemotherapy nor radiotherapy preoperatively. DNA was extracted and PCR was used to test the homozygous deletion of p15 exon 2 (p15E2) and p16 exon 2 (p16E2). RNA was extracted, cDNA was synthesized by reverse transcription, and the expression of STK15 gene was tested by PCR with beta-actin as inner control. At the same time the expression of STK15 in human LSCC Hep-2 cell line was tested. The ratio of ADV (average density value) of STK15 gene to the ADV of beta-actin gene was calculated.The rate of p15E2 deletion was 12% (6/50) and that of p16E2 was 14% (7/50). The p15E2 and p16E2 codeletion rate was 6% (3/50). In 34 of the 50 cases (68%) the expression of STK15 gene in tumor tissue was higher than that of the paired surrounding normal tissue with a significant difference. The ratio of ADV of STK15 gene to ADV of beta-actin gene was 1.03 +/- 0.30 in the cancer tissue, and 0.89 +/- 0.22 in the paired normal tissue with a significant difference (t = 4.333, P0.01). The expression of STK15 gene was higher than that of beta-actin in Hep-2 cell line.The homozygous deletion of p15E2 and p16E2 and overexpression of STK15 gene may play a role in the oncogenesis and malignant progression of laryngeal squamous cell carcinoma.