Cep68 can be regulated by Nek2 and SCF complex.

Centrosome cohesion maintains centrosomes in close proximity until mitosis, when cell cycle-dependent regulatory signaling events dissolve cohesion and promote centrosome separation in preparation for bipolar spindle assembly at mitosis. Cohesion is regulated by the antagonistic activities of the mitotic NIMA-related kinase 2 (Nek2), protein phosphatase 1, the cohesion fiber components rootletin, centrosomal Nek2-associated protein 1 (C-Nap1) and Cep68. The centrosomal protein Cep68 is essential for centrosome cohesion and dissociates from centrosomes at the onset of mitosis. Here, our cell line studies show the C-terminal 300-400 amino acids of Cep68 are necessary to localize Cep68 to interphase centrosomes while C-terminal 400-500 amino acids might regulate Cep68 dissociation from centrosomes at mitotic onset. In addition, Nek2 was demonstrated to phosphorylate Cep68 in vivo and this phosphorylation appears to promote Cep68 degradation in mitosis. We further show that the SCF complex destroys Cep68 at mitosis through recognition by the beta-Trcp F box component of SCF. Together, the findings provide a new insight into the control of centrosome separation by Cep68 during mitosis.

Loss of heterozygosity at chromosomes 1p35-pter, 4q, and 18q and protein expression differences between adenocarcinomas of the distal stomach and gastric cardia.

Loss of heterozygosity of 1p35-pter, 4q, and 18q is frequent in gastric carcinoma, suggesting that these regions harbor tumor suppressor genes. However, the differences in these genetic alterations between adenocarcinoma of the gastric cardia and adenocarcinoma of the distal stomach remain unclear. In this study, loss of heterozygosity at chromosomes 1p35-pter, 4q, and 18q were analyzed in adenocarcinoma of the gastric cardia and adenocarcinoma of the distal stomach samples acquired by laser capture microdissection. The expression of several tumor suppressor gene proteins, runt-related transcription factor 3 (1p36), annexin A10 (4q33), SMAD family member 4 (18q21.1), and deleted in colorectal carcinoma (18q21.3), was evaluated immunohistochemically. The adenocarcinoma of the distal stomach and adenocarcinoma of the gastric cardia lesions had a similar trend in total deletion frequency for chromosomes 1p35-pter (36.5% for adenocarcinoma of the distal stomach and 32.5% for adenocarcinoma of the gastric cardia), 4q (42.3% for adenocarcinoma of the distal stomach and 47.5% for adenocarcinoma of the gastric cardia), and 18q (38.5% for adenocarcinoma of the distal stomach and 45% for adenocarcinoma of the gastric cardia). However, loss of heterozygosity patterns were clearly different in the 2 adenocarcinomas. Deletion mapping indicated that 4q32.2-4q34.3, 18q21.2-21.31, 18q22.3-23, and 1p35.2-1p36.13 were involved in adenocarcinoma of the distal stomach, whereas 4q13.3-4q22.3, 4q31.21-4q32.2, 18q21.31-18q22.1, and 1p35.2-1p36.13 were involved in adenocarcinoma of the gastric cardia. Expression of ANXA10 (P = .038), SMAD family member 4 (P = .028), and deleted in colorectal carcinoma (P = .004) was less common in adenocarcinoma of the distal stomach than in adenocarcinoma of the gastric cardia. Expression of runt-related transcription factor 3 (P = .795) showed no significant difference in the 2 tumors. The tumors differed in the profile of genetic alterations and protein expression of these well-known tumor suppressor genes. The deleted regions defined in this study may harbor tumor suppressor genes relevant to adenocarcinoma of the gastric cardia.

Identification of the keratin 9 (KRT9) N161S mutation in a Chinese kindred with epidermolytic palmoplantar keratoderma.

We present a family from Northeast China affected by epidermolytic palmoplantar keratoderma (EPPK) in which we confirmed the presence of the N161S mutation as the result of a 548A>G transition in exon1 of the keratin 9 gene. Genomic DNA from peripheral blood of all available members in this family was used for amplification of exon 1 of KRT9 by polymerase chain reaction. The mutation was detected by direct sequence analysis and identified by restriction endonuclease DdeI digestion. The finding of the same mutation in all available patients, together with the previous reports of the disease, strongly suggested that position 161 of the KRT9 gene also represents a mutation "hotspot" for EPPK. Our result is an important contribution to the investigation of the genotype/phenotype correlation and affords molecular genetic knowledge for future clinical diagnosis and gene therapy of EPPK.

Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed therapeutics.

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P < 0.001, n = 81). However, no significant correlation of EGFR expression with disease-free survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from the normal condition through disease progression using in vitro, xenograft, and matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting the EGFR family of proteins in breast cancer.

Molecular prenatal diagnosis for hereditary distal arthrogryposis type 2B.

Autosomal dominant distal arthrogryposes (DAs) are a group of muscle diseases characterized by congenital contractures of the limbs. Currently, prenatal diagnosis of DAs depends upon ultrasound examination during late gestation. Recently, five genes encoding fast switch proteins located at 9p13.2, 11p15.5 and 17q13.1 were identified. These included TPM2, TNNI2/TNNT3, and MYH3/MYH8. Last year, we discovered a novel heterozygous mutation c.523_525delAAG (p.K175del) in the TNNI2 gene, which encodes the isoform of troponinI, in a seven-generation Chinese family affected with distal arthrogryposis type 2B (DA2B). Here, we report the molecular prenatal diagnosis of 3 high-risk fetuses of two women in the family by two-point linkage inferential analysis and deletion detection of the TNNI2 gene with chorionic villus sampling (CVS) or amniocentesis. To our knowledge, this is the first description of molecular prenatal diagnosis for DAs.

[Loss of heterozygosity at chromosome 8p21-p23 in adenocarcinoma of gastric cardia].

Chromosome 8p21-p23 harbors tumor suppressor gene(s) implicated in multiple types of cancers. To investigate the involvement of the gene(s) in the carcinogenesis of adenocarcinoma of gastric cardia, loss of heterozygosity (LOH) for microsatellite markers at chromosome 8p21-p23 was examined. Laser capture microdissection (LCM) was used to obtain homogeneous tumor cells from 19 surgical specimens. Subsequently, genomic DNA extracted from the LCM-captured cells was amplified by multiple displacement amplification. Each tumor was assessed for allelic loss using 13 microsatellite markers. An overall LOH frequency of 63.2% (12/19) was observed and the LOH frequency for individual markers varied from 25% to 55.6%. One common deleted region of about 1.2 Mb (8p22GGAA-8p22ATCT) was defined. Our data indicated that the tumor suppressor gene at chromosome 8p22 might play an important role in the development of adenocarcinoma of gastric cardia.

Preliminary analysis of CDK2 sequence and its nuclear import.

We constructed the plasmids encoding enhanced green fluorescent protein (EGFP)-tagged wild type cyclin-dependent kinase 2(CDK2) (pEGFP-CDK2) and CDK2 deletion mutants (pEGFP-CDK2N and pEGFP-CDK2C, lacking the last C-terminal and the first N-terminal 97 amino acids of CDK2, respectively) and transfected them into HeLa cell line and CHO cell line. After synchronization, green fluorescent signals were detected mainly in nucleus of the cells transfected with pEGFP-CDK2 and predominantly in cytoplasm of the cells transfected with the two mutant CDK2 constructs. Our results suggested that there were no nuclear-import signals in CDK2 and that CDK2 nuclear import might be mediated by association with other proteins through the three-dimensional structure formed by amino acids including those from the N- and C-terminal regions of CDK2.

Effect of cyclin G2 on proliferative ability of SGC-7901 cell.

AIM: To study the effect of cyclin G2 on proliferation of gastric adenocarcinoma cell line-SGC-7901 cell in vitro. METHODS: By use of cation lipofectamine transfection reagent, the pIRES-G2 and pIRESneo plasmids were transferred into SGC-7901cell line. Anticlones were selected by G418. Positive clones were observed and counted using Giemsa staining. Cell proliferative ability was assayed by MTT. RESULTS: (1) The clone number of pIRES-G2 group decreased, clone volume reduced. The number of cell clones in pIRESneo group was 87+/-3, that of pIRES-G2 group was 53+/-4, occupying 60.1% of pIRESneo group, there was significant difference obviously (P<0.01, t=15.45). (2) The average absorbance of clone cell obtained by stable transfection of pIRES-G2 at 570 nm was 1.6966+/-0.2125, the average absorbance of clone cell obtained by stable transfection of pIRESneo at 570 nm was 2.1182+/-0.3675, there was significant difference between them (P<0.01, t=3.412). CONCLUSION: Cyclin G2 can inhibit SGC-7901cell proliferative ability obviously, it may be a negative regulator in cell cycle regulation.