Establishment of partial gene expression map of 7q32 in nasopharyngeal carcinoma and primary culture normal nasopharyngeal epithelial cells

OBJECTIVE: To establish partial gene expression map of 7q32 in nasopharyngeal carcinoma (NPC) cell line, tissues and primary culture normal nasopharyngeal epithelial cells. METHODS: We detected the expression of 20 ESTs at 7q32 in NPC cell line HNE1,13 NPC biopsies and primary culture normal nasopharyngeal epithelial cells using differential RT PCR and Northern hybridization. RESULTS: 8 ESTs (AA188181, AA13079,N27556, AA031919, N22721, H20825, T91284, AA001936) expressed equally in both of HNE1 and primary culture normal nasopharyngeal epithelial cells; 7 ESTs (T64215, AA025822, R60014,R80002,H06688, R60192,R95096) expressed in neither of them; 3 ESTs (H19830,W72688,AA130630) overexpressed in HNE1 ; and 2 ESTs (AA070437, H90882) overexpressed in primary culture normal nasopharyngeal epithelial cells. W72688 and H19830 each overexpressed in 77%(10/13) of NPC biopsies; AA070437 down-expressed in 30.7% of NPC biopsies. CONCLUSION: Partial gene expression map of 7q32 in nasopharyngeal carcinoma cell line ,tissues and primary culture normal nasopharyngeal epithelial cells has been established. The up-regulation of W72688, H19830 and down-regulation of AA070437 may be related to the occurrence of NPC.     

Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential

In this study we have sought to characterize a committed myeloid progenitor cell line in an attempt to isolate general factors that may promote differentiation. We used cDNA representational difference analysis (RDA), which allows analysis of differential gene expression, to compare EML and EPRO cells. We have isolated nine differentially expressed cDNA fragments as confirmed by slot blot, Northern, and PCR analysis. Three of nine sequences appear to be novel whereas the identity of the remaining fragments suggested that the EPRO cell line is multipotent. Among the isolated sequences were eosinophilic, monocytic, and neutrophilic specific genes. Therefore, we tested the ability of EPRO cells to differentiate along multiple myeloid lineages and found that EPRO cells exhibited morphologic maturation into either monocyte/macrophages or neutrophils, but not eosinophils. Furthermore, when EPRO cells were exposed to ATRA, neutrophil specific genes were induced, whereas monocytic markers were induced by phorbol ester treatment. This study highlights the use of cDNA RDA in conjunction with the EML/EPRO cell line to isolate markers associated with macrophage and neutrophil differentiation and establishes the usefulness of this system in the search for factors involved in myeloid commitment.     

Detection of differential gene expression in human osteoblastic cells by non-radioactive RNA arbitrarily primed PCR

The aim of the present study was to detect differentially expressed genes in the human osteoblast-like osteosarcoma cell line SaOS-2 using non-radioactive RNA fingerprinting (RNA arbitrarily primed polymerase chain reaction, RAP-PCR). RNA was isolated at different time points from SaOS-2 cells grown with and without dexamethasone (DEX). By RAP-PCR we detected changes in band patterns of cells treated with DEX compared with untreated cells. PCR fragments further characterized and sequences from three of these gave perfect matches to the coding sequences of the human nucleophosmin gene B23, cDNA clone 4_c6 from P1 H25 and the human TRA1 gene, respectively. differential regulation of these genes in DEX-stimulated SaOS-2 cells could be demonstrated by RT-PCR.     

Metastasizing placental site trophoblastic tumor: immunohistochemical and DNA analysis. 2 case reports and a review of the literature

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.     

Infrequent MDM2 gene amplification and absence of gross WAF1 gene alterations in nasopharyngeal carcinoma

We have investigated the possible involvement of MDM2 and WAF1 gene alterations in the development of nasopharyngeal carcinoma (NPC). MDM2 and WAF1 were analysed in 46 primary NPCs by Southern blot analysis. Forty-five tumours showed a normal EcoRI hybridisation pattern and hybridisation intensity with a human MDM2 cDNA probe. One tumour had more intense normal size MDM2 hybridising bands. Densitometric scanning revealed a 10-12-fold MDM2 gene amplification, as compared with human placenta DNA. All 46 tumours showed normal size WAF1 EcoRI bands that hybridised with normal intensity. This is the first demonstration of MDM2 gene amplification in NPC. Nonetheless, our analysis indicates that gross structural alterations of the MDM2 and WAF1 genes are infrequent events in the genesis of NPC.     

Rapid isolation of cell-type-specific protein tyrosine kinases by degenerate polymerase chain reaction combined with differential hybridization technique

To identify protein tyrosine kinase (PTK) genes preferentially expressed in renal cell carcinoma cell line, we screened a PTK-cDNA-enriched library constructed from RNA of an renal cell carcinoma cell line with a PTK probe, each produced from renal cell carcinoma, gastric cancer or esophageal cancer cell lines by degenerate polymerase chain reaction. Two cDNA fragments of PTK genes, FRK and FLT-3, were isolated from the PTK-cDNA-enriched library of the renal cell carcinoma cell line by differential hybridization technique. The FRK cDNA clone represented 15.8% of the PTK-cDNA-enriched library from the renal cell carcinoma cell line, while the FLT-3 cDNA clone was 2.8% of the same library. Both of the two PTK genes were expressed preferentially in renal cell carcinoma cell lines. This method, described here, is useful for the rapid isolation of PTK cDNA fragments, including a low abundant cDNA, preferentially expressed in a specific cell line.     

The success of locoregional, low-dose recombinant interleukin-2 therapy in tumor-bearing mice is dependent on the time of rIL-2 administration

The most common chromosomal aberrations in myelodysplastic syndromes (MDS) are complete or partial loss of chromosomes 5 and 7, and trisomy 8. To identify genes important in the pathogenesis of this disease that could be associated with these gross chromosomal defects, we have employed the differential display PCR (DDPCR) procedure developed by Liang and Pardee. This method allows simultaneous comparison of several cDNA sources for the presence of differentially expressed genes. Polymorphonuclear cells (PMNs) from two MDS patients, containing a 5q deletion or a trisomy 8, and three healthy controls were used. Initial screening resulted in the identification of five and three partial cDNA sequences, respectively that were either differentially expressed in both patient samples or in individual patients, as compared with the controls. The authenticity of aberrant expression was verified by reanalyzing the same primer combinations on newly prepared cDNA. Differential expression of the three remaining fragments was subsequently checked on a larger panel of MDS patients, using amplicon-specific primer sets. These were obtained by cloning and sequencing of the fragments. For one partial cDNA (DC3), the original expression pattern, i.e., decreased expression in individual MDS patients, was confirmed. These results demonstrate the utility of the DDPCR procedure to isolate differentially expressed sequences in primary patient samples where the availability of cells is a limiting factor.     

Applicability of nonsyngeneic cell models for screening of genes in monogenetic diseases via differential display technique

Conventional subtraction library techniques or DNA-transfection studies are standard techniques applied for identification and isolation of genes relevant in monogenetic diseases like Fanconi anemia (FA). The differential display technique (DDT) was developed to compare mRNA expression between a mutant cell line and its syngeneic control and allows comparison of almost all mRNA species within a short time. However, for identification of genes relevant in monogenetic diseases, no syngeneic cell model is available. In this report, we show that the use of nonsyngeneic diploid human fibroblasts does not increase the number of differentially displayed bands due to diversity of untranslated regions. cDNA bands with a length of up to 1000 bp were obtained and applied to DDT. After screening of about 13000 cDNA bands, only 0.5% were found to be differentially expressed between FA and control cells. Finally, three mRNAs were cloned and verified in Northern blot experiments to be differentially expressed in FA fibroblasts. The low number of differentially displayed cDNA bands in DDT indicates the usefulness of this statistical, molecular approach for identification of multiple genes dysregulated in gene regulation cascades potentially relevant for cell cycle disturbances.     

Fasting suppresses pulsatile luteinizing hormone (LH) secretion and enhances orderliness of LH release in young but not older men

BACKGROUND: Studies identifying genes that are differentially expressed following induction of acute ischemic injury have been useful in delineating the pathophysiology of acute renal failure. METHODS: A differential cDNA library screening technique was used to identify genes that are differentially expressed in rat kidney following induction of acute ischemic renal injury. RESULTS: Levels of mRNA with a high homology to that coding for Siva, a human proapoptotic protein, were increased approximately 4.5-fold in kidneys obtained from rats within 12 hours following ischemia, compared to kidneys from sham-operated rats. A partial cDNA sequence for the rat protein (rat Siva) was determined that overlaps 92% of the human open reading frame. The cDNA sequence predicts a protein 177 amino acids in length with 76% homology to human Siva. Levels of rat Siva in kidneys were elevated at one, five and seven days post-ischemia were not different from those in kidneys from sham-operated controls. In situ hybridization demonstrated that rat Siva mRNA was expressed in cells lining damaged sections in the S3 segment of the proximal tubule at 12 hours and one day post-ischemia. At five and seven days, Siva mRNA was located in epithelial cells of regenerating tubules including in papillary proliferations. TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells colocalized with cells containing Siva mRNA. CD27, the receptor for Siva was localized by immunohistochemistry to sloughed cells in the lumens of damaged S3 segments at 12 hours post-ischemia and to cells within papillary proliferations at five days post-injury. CONCLUSIONS: Siva that is produced within the kidney could be a mediator of apoptosis post-ischemia via an interaction with CD27.     

对比分析黑斑息肉综合征合并大肠癌组织肿瘤基因

Objective: The aim of the study was to screen the differentially expressed genes of Peutz-Jeghers syndrome (PJS) and colorectal carcinoma (CRC). Methods: This study used cDNA microarray to comparatively analyze the gene expression profiles of 4 cases of PJS combined with colorectal adenocarcinoma vs. normal mucosae. The cDNA microarray contained 8064 human genes, and then using RT-PCR to test three genes of all. Results: The experimental data showed that fourteen genes were differentially expressed, which were up-regulated in PJS. Fifty-one genes expressions were altered in CRCs, of which 32 were up-regulated, as compared to the normal mucosae. In addition, 5 genes were similarly altered in both PJS and CRCs. RT-PCR analyses confirmed the cDNA microarray data for three of those genes: LATS2, APC and MADH4. Conclusion: LCN2, USP4, GRO3, HYAL1 and APC - these differentially expressed genes likely represent biomarkers for early detection of CRC and may be potential therapeutic targets.