OBJECTIVE: To investigate the profile and potential significance of PTEN and NBS1 mutations among patients with familial or at early onset breast cancer in Hunan province.
 METHODS: A total of 131 breast cancer patients with familial history or suffered from breast cancer at the age of less than 35 years old were included in this study. A comprehensive phosphatase and tensin homolog (PTEN) and nibrin (NBS1) mutation analysis was performed through denaturing high performance liquid chromatography (DHPLC) and subsequent DNA direct sequencing.
 RESULTS: Among 131 patients, a reported mutation IVS4+109insTCTTA in PTEN gene were identified in two patients. The mutation frequency of IVS4+109insTCTTA was 1.15%. Two mutations in PTEN gene, 225 A>C (Thr 160 Pro) and IVS5+13T>C, was firstly discovered. Another reported missense mutation was rs121909229 G>A (Arg 130 Gln). Three mutations were detected in NBS1 gene, of which IVS6+43A>G and IVS6+127A>G were firstly discovered and another reported synonymous mutations was rs1805794 G>C (Glu 185 Gln).
 CONCLUSION The novel mutations in PTEN and NBS1 might be specific to the familial and early-onset breast cancer of Chinese Hunan population.
Adult ; Asian Continental Ancestry Group ; Breast Neoplasms ; genetics ; Cell Cycle Proteins ; genetics ; China ; DNA Mutational Analysis ; Female ; Humans ; Mutation ; Nuclear Proteins ; genetics ; PTEN Phosphohydrolase ; genetics

BACKGROUNDNucleostemin is essential for the proliferation and survival of stem and cancer cells, but it is unknown whether this newly identified molecule is involved in prostate cancer pathogenesis.

METHODSTotal RNA and protein were extracted from prostate cancer tissues and PC-3, LNCap and DU145 cell lines. The nucleostemin mRNA and protein expression were measured by RT-PCR and Western blot. Immunohistochemistry was also used to detect the nucleostemin protein expression in prostate cancer tissues and PC-3 cells. A nucleostemin specific, short hairpin RNA, expression plasmid was used to transfect PC-3 cells. The changes of nucleostemin gene were detected and the proliferative capacity of the cells was determined.

RESULTSNucleostemin was highly expressed in prostate cancer tissues and cell lines. Nucleostemin expression level in the silencer group PC-3 cells remarkably reduced. The proliferation rate of silencer group PC-3 cells decreased and the percentage of G1 stage cells increased. The neoplasm forming capacity in nude mice of the silencer group PC-3 cells decreased significantly.

CONCLUSIONSNucleostemin is highly expressed in prostate cancer tissues and cell lines. The proliferative capacity of PC-3 cells is remarkably reduced after silencing nucleostemin gene expression.

Animals ; Apoptosis ; Carrier Proteins ; analysis ; genetics ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; GTP-Binding Proteins ; Humans ; Male ; Mice ; Nuclear Proteins ; analysis ; genetics ; physiology ; Prostatic Neoplasms ; genetics ; pathology ; RNA, Messenger ; analysis

OBJECTIVETo study the influence of human cytomegalovirus (HCMV) infection on cell cycle and the expression of replication licensing factor Cdt1 in human embryonic lung fibroblastic (HEL) cells and to explore the pathogenesis of HCMV infection.

METHODSHEL cells were synchronized in the G0/G1 phase by the serum starvation method. The synchronized HEL cells were infected with HCMV, and those that were not subjected to HCMV infection were used as the control group. The HEL cells were harvested at 12, 24, 48, 72 and 96 hrs of HCMV infection. The cell cycle of HEL cells was detected by the flow cytometry. The expression of Cdt1 mRNA in HEL cells was determined by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe cells in the G1 phase in the control group was significantly more than in the HCMV-infected group 12 and 24 hrs after infection (P < 0.01). The expression of Cdt1 mRNA in the HCMV-infected group was significantly lower 12 and 24 hrs after infection but increased significantly 48 hrs after infection compared with the control group (P < 0.05). The expression of Cdt1 mRNA reached a peak at 12 hrs of infection in the control group, but at 48 hrs of infection in the HCMV-infected group, which markedly lagged behind the control group.

CONCLUSIONSHCMV infection arrests the cell cycle of HEL cells at the G1 phase. HCMV infection makes Cdt1 expression delay. HCMV infection can interfere cell cycle of HEL cells possibly through affecting the expression of Cdt1.

Cell Cycle ; Cell Cycle Proteins ; genetics ; Cells, Cultured ; Cytomegalovirus ; pathogenicity ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung ; cytology ; metabolism ; RNA, Messenger ; analysis

OBJECTIVETo analyze the clinical features and genotype in a 8-year-old boy with dyskeratosis congenita (DC).

METHODSWe reviewed the clinical data of the case and amplified 7 DC-related genes (including DKC1,TERT,TERC,TINF2,NOP10, NHP2 and WRAP53) using polymerase chain reaction for DNA sequence analysis to identify the abnormal exons.

RESULTSDNA sequence analysis showed a c.85-15T>C mutation in DKC1 gene of the patient. His mother was a carrier of the mutated gene and presented with partial clinical features such as abnormal nails.

CONCLUSIONThe mutation of c.85-15T>C in DKC1 gene was reported for the first time in China. The diagnosis of DC should be considered if a young patient presents with mucocutaneous abnormalities, bone marrow failure, cancer susceptibility and a family history of cancer. Early genetic tests can improve the diagnosis rates and reduce misdiagnosis and missed diagnosis.

Cell Cycle Proteins ; genetics ; Child ; China ; Dyskeratosis Congenita ; genetics ; pathology ; Exons ; Genotype ; Humans ; Male ; Mutation ; Nuclear Proteins ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo study the relationship between hRad17 mRNA expression and clinicopathologic factors and lymph node metastasis of gastric cancer, and to assess the significance of predicting the extent of lymph node metastasis and prognosis.

METHODShRad17 mRNA expression was examined in matched primary lesions, normal gastric mucosa and lymph node metastatic lesions among 52 gastric cancer patients by reverse transcription polymerase chain reaction (RT-PCR), polyacrylamide gel electrophoresis (PAGE) and silver stain with the relation between hRad17 mRNA expression and clinicopathologic factors analyzed. At the same time, hRad17 mRNA expressions in 5 gastric benign lesions and SGC7901 gastric carcinoma cell lines were also examined.

RESULTSThe primary tumor samples (88.4% positive) showed a significantly higher level of hRad17 expression compared with matched normal tissue (76.9% positive) (P = 0.014), so did the lymph node metastatic samples (94.2% positive) (P = 0.001). The hRad17 mRNA expression showed a low level in benign lesions, but very high in SGC7901 cell line. The hRad17 mRNA expression showed a higher level in patients with the number of lymph node metastasis above 15 than below 15 (P = 0.02), so did the diffused growth than the mass-like growth (P = 0.04).

CONCLUSIONThe method of PAGE and silver stain can improve the sensitivity of RT-PCR. The degree of lymph node metastasis and invasiveness of carcinoma cells are more serious in cases with hRad17 mRNA overexpression, and extensive lymph node dissection should be carried out for these patients. Examination of hRad17 expression by RT-PCR before surgery is indicated to arrive at an optimum treatment scheme and to estimate the prognosis.

Cell Cycle Proteins ; genetics ; Gastric Mucosa ; metabolism ; Humans ; Lymphatic Metastasis ; RNA, Messenger ; analysis ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVE: To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis. METHODS: NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR). RESULTS: Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%. CONCLUSION HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.
Cell Cycle ; Cell Line ; Gene Expression Profiling ; Hepacivirus ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptional Activation ; Viral Nonstructural Proteins ; genetics

Cornelia de Lange syndrome (CdLS) is a clinically and genetically heterogeneous congenital anomaly. Mutations in the NIPBL gene account for a half of the affected individuals. We describe a family with CdLS carrying a novel pathogenic variant of the SMC1A gene identified by exome sequencing. The proband was a 3-yr-old boy presenting with a developmental delay. He had distinctive facial features without major structural anomalies and tested negative for the NIPBL gene. His younger sister, mother, and maternal grandmother presented with mild mental retardation. By exome sequencing of the proband, a novel SMC1A variant, c.3178G>A, was identified, which was expected to cause an amino acid substitution (p.Glu1060Lys) in the highly conserved coiled-coil domain of the SMC1A protein. Sanger sequencing confirmed that the three female relatives with mental retardation also carry this variant. Our results reveal that SMC1A gene defects are associated with milder phenotypes of CdLS. Furthermore, we showed that exome sequencing could be a useful tool to identify pathogenic variants in patients with CdLS.
Asian Continental Ancestry Group/genetics ; Base Sequence ; Cell Cycle Proteins/*genetics ; Child, Preschool ; Chromosomal Proteins, Non-Histone/*genetics ; DNA ; DNA Mutational Analysis ; De Lange Syndrome/diagnosis/*genetics ; Female ; Heterozygote ; Humans ; Male ; Pedigree ; Phenotype ; Polymorphism, Single Nucleotide ; Proteins/genetics ; Republic of Korea

BACKGROUNDBmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.

METHODSA transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain.

RESULTSK562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.

CONCLUSIONThe antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Humans ; K562 Cells ; Nuclear Proteins ; antagonists & inhibitors ; genetics ; Plasmids ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; RNA, Antisense ; physiology ; Repressor Proteins ; antagonists & inhibitors ; genetics

OBJECTIVETo investigate the relationship between expressions of ING1 gene and genes of human telomerase reverse transcriptase (hTERT) and telomerase-associated protein 1 (hTP1) in human gliomas.

METHODSThe expressions of ING1 mRNA and p33(ING1) protein, hTERT mRNA and protein, and hTP1 mRNA and protein in seventy human glioma specimens with different malignant grades were studied using in situ hybridization and immunohistochemistry.

RESULTSAll of the 70 gliomas collected expressed hTP1 mRNA and protein and among them, 62 (88.6%) and 58 (82.9%) out of 70 expressed hTERT mRNA and protein respectively. The quantities of the four kinds of positive cells were correlated positively with one another (r = 0.758 - 0.882, P < 0.000 5), and all of them were significantly fewer in gliomas of WHO grade I - II than in grade III gliomas and the most in grade IV gliomas (P < 0.05 approximately 0.01). 66 (94.3%) and 62 (88.6%) out of 70 gliomas expressed ING1 mRNA and p33(ING1) protein respectively. The quantities of their positive cells were also correlated positively with each other (r = 0.831, P < 0.000 5), but the positive cells were more in gliomas of WHO grade I - II than in grade III gliomas and the fewest in grade IV gliomas (P < 0.01). The quantities of positive cells of ING1 mRNA and p33(ING1) protein were correlated negatively with those of hTERT mRNA and protein as well as hTP1 mRNA and protein respectively (r = -0.211 to -0.384, P < 0.05 approximately 0.001).

CONCLUSIONSThe results suggest that all of the parameters concerned are valuable in evaluating the biological behavior of gliomas. In glioma cells, overexpressions of hTERT and hTP1 genes might be significant in inhibiting the expression of ING1 gene. The abnormal expressions of the three genes play possibly the important roles in the development and malignant progression of gliomas.

Adolescent ; Adult ; Aged ; Carrier Proteins ; analysis ; genetics ; Cell Cycle Proteins ; DNA-Binding Proteins ; Female ; Genes, Tumor Suppressor ; Glioma ; genetics ; metabolism ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; Male ; Membrane Transport Proteins ; Middle Aged ; Nuclear Proteins ; Proteins ; genetics ; RNA, Messenger ; analysis ; Telomerase ; analysis ; genetics ; Tumor Suppressor Proteins

OBJECTIVEBoth tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene.

METHODSAfter identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry.

RESULTSNeither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene.

CONCLUSIONp15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; Cell Cycle Proteins ; genetics ; Cell Division ; Cyclin-Dependent Kinase Inhibitor p15 ; Genes, Retinoblastoma ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Liver Neoplasms ; genetics ; pathology ; RNA, Messenger ; analysis ; Tumor Suppressor Proteins ; genetics