Kang Ying

Discovery and analysis of hepatocellular carcinoma genes using cDNA microarrays.

Abstract Purpose. Microarray analysis on a genomic scale was used to profile changes in gene expression accompanying hepatocellular carcinoma. Methods. Gene expression profiles of liver tissues from twelve hepatocellular carcinoma samples relative to the gene expression profile of the normal liver tissue were analyzed using 4096 chips and 12800 chips. The results of microarray experiments were verified by the Northern blot technique. Results. A group of 1,820 genes with altered expression were identified in more than 50% of the patients examined. This highly concordant expression profile included human genes encoding proteins involved in the function of peroxisomes, serum control, polycyclic aromatic hydrocarbon carcinogenesis, cell growth and differentiation, metastasis, the function of the immune system, apoptosis, and remodeling of the cytoskeleton. Conclusions. The newly identified genes afford a quantitative view of the changes that accompany liver cancer at the genomic level, enable deeper insights into the molecular basis of disease, and provide an extensive list of potential early-onset molecular markers for improved diagnosis.

Cloning and characterization of a human lysyl oxidase-like 3 gene (hLOXL3)

Using the PCR primers generated from human expressed sequence tag (EST), the cDNA of lysyl oxidase-like gene 3 (LOXL3), a new member of human lysyl oxidases gene family, was cloned from the human fetal brain mRNA. The predicted amino acid sequence of the hLOXL3 gene was highly homologous to mLOR2. Bioinformatics analysis shows that hLOXL3 protein is also a member of the scavenger receptor cysteine-rich family, which contains a 25 amino acids signal peptide. The hLOXL3 gene was mapped to human 2p13 locus by BLAST search and at least 14 exons were found. Expression of the hLOXL3 gene was detected in several human tissues and especially high in spleen and testis.

Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray

Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL‐60 promyeloid leukemia cells exposed to cantharidin. Cantharidin‐treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c‐myc, GTPase) or show tumor‐specific expression (e.g., phosphatidylinositol 3‐kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth‐inhibitory proteins (e.g., BTG2, MCP‐3) as well as proapoptotic genes (e.g., ATL‐derived PMA‐responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin‐mediated cytotoxicity. We also found that exposure of HL‐60 cells to cantharidin resulted in the decreased expression of multidrug resistance‐associated protein genes (e.g., ABCA3, MOAT‐B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL‐3, N‐formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin.

Cloning and Identification of a Novel cDNA Coding Thioredoxin-Related Transmembrane Protein 2

Thioredoxin plays an important role in various cellular processes through redox regulation. Here we report the molecular cloning and characterization of one member of the thioredoxin superfamily, designated as TMX2. The TMX2 cDNA consists of 1644 nucleotides and contains an open reading frame encoding a protein of 372 amino acids with a predicted molecular mass of 42.5 kDa and an isoelectric point of 8.94 . The TMX2 protein may possess an N-terminal signal peptide, a potential transmembrane domain, an Myb DNA-binding domain repeat signature, a thioredoxin consensus pattern, an endoplasmic reticulum (ER) membrane retention signal (KKXX-like motif), and a dileucine motif in the tail. Northern blot analysis shows it is widely expressed in human tissues.

Identification and characterization of AGTRAP, a human homolog of murine Angiotensin II Receptor-Associated Protein (Agtrap) ☆

Several classes of cytoplasmic proteins have been found to interact specifically with the carboxyl-terminal cytoplasmic region of the angiotensin II type 1 (AT(1)) receptor to regulate different aspects of AT(1) receptor physiology. The murine Angiotensin II Receptor-Associated Protein (Agtrap) is a new member of them. We have recently cloned a new human gene cDNA that codes for a homolog of the murine Agtrap protein from a human fetal brain cDNA library. The deduced polypeptide product of the cDNA is 22 kDa in size, and its DNA and amino acid sequences are 85 and 77% identical to those of the mouse Agtrap gene, respectively. Hence we have named it the human Angiotensin II Receptor-Associated Protein (AGTRAP) gene. The mRNA of AGTRAP was most abundantly expressed in kidney, heart, pancreas and thyroid. Using the yeast two-hybrid screening of a human fetal brain cDNA library, we have identified a new interaction partner of the human AGTRAP protein, RACK1 (Receptor of Activated Protein C Kinase). The AGTRAP-RACK1 interaction was confirmed by GST fusion protein pull-down assays, co-immunoprecipitation and surface plasmon resonance. We suggest that the AGTRAP-RACK1 interaction may help to recruit signaling complex to the AT(1) receptor to affect AT(1) receptor signaling.

Cloning and expression pattern of the human NDRG3 gene

We report the cloning and expression pattern of a novel N-myc downstream-regulated gene 3 (NDRG3), located on human chromosome 20q11.21-11.23. The NDRG3 cDNA is 2588 base pair in length, encoding a 363 amino acid polypeptide highly related to mouse Ndr3 protein. Northern blot reveals that NDRG3 is highly expressed in testis, prostate and ovary. By in situ hybridization, the NDRG3 mRNA was localized to the outer layers of seminiferous epithelium, indicating that it may play a role in spermatogenesis.

Cloning and characterization of a novel human transcription factor AP-2β like gene (TFAP2BL1)

The AP-2 transcription factor has been shown to play an important role in development, morphogenesis, apoptosis, cell-cycle control and has also been implicated in mammary oncogenesis. Here we report the cloning and characterization of a novel human transcription factor AP-2 like gene (TFAP2BL1), which is located on human chromosome 6p12.1-21.1. The TFAP2BL1 cDNA is 2076 base pairs in length, encoding a 452-amino acid polypeptide related to human Ap-2protein. TFAP2BL1 gene has significantly high homology to transcription factor AP-2 gene of human, mouse, chicken, sheep, fruit fly, and C. elegans at amino acid level. RT-PCR analysis shows its relatively high expression level in adult thymus, prostate, small intestine, skeletal muscle, placenta, brain, and testis tissues.

Cloning and Expression Pattern of a Spermatogenesis-Related Gene, BEX1, Mapped to Chromosome Xq22

Through screening a human fetal brain cDNA library, a cDNA similar to the mouse Bex1 was isolated. This new gene was named brain expressed X-linked protein 1 (BEX1). Northern blot analysis revealed a 1.0 kb transcript highly expressed in brain, pancreas, testis, and ovary, with lower levels present in heart, placenta, liver, kidney, spleen, thymus, prostate, small intestine, colon ( no mucus), thyroid, spinal cord, and adrenal gland. No hybridization signal was seen in lung, skeletal muscle, peripheral blood leukocyte, stomach, lymph node, trachea, and bone marrow. The BEX1 gene was localized to chromosome band Xq22 between markers between DXS990 and DXS1059 by screening Stanford radiation hybrid G3 panels. In situ hybridization of mouse testis using BEX1 as a probe detected the signal in the pachytene spermatocytes and spermatids but not in spermatogonia. Furthermore, it was not detected at 6, 9, and 12 days postpartum, was present in low amount on Days 15 and 18 and its expression increased sharply after the initiation of puberty (about 21 days) in mouse testis.

Identification of a novel human angiopoietin-like gene expressed mainly in heart

AbstractThe angiopoietins are an important family of growth factors specific for vascular endothelium. Most of them bind to the TIE2 receptor and are related to regulation of angiogenesis. During large-scale DNA sequencing of the human fetal brain cDNA library, we cloned a novel human angiopoietin-like cDNA and termed it human angiopoietin-like 5 (ANGPTL5). Like other members of the angiopoietin family, ANGPTL5-deduced protein also has an N-terminal cleavable signal peptide, a predicted coiled-coil domain, and a fibrinogen-like domain. The search against the human genome database indicated that ANGPTL5 maps to 11q22. Expression analysis of ANGPTL5 shows that it is mainly expressed in adult human heart.

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.