A large number of data derived from molecular analyses support the hypothesis that human cancer is a genetic disease and a distinct subset of genes have been found to be genetically changed in most tumors. Molecular alterations in pancreatic cancer include: (1) oncogenes such as K-ras, c-myc, c-fos, and c-erbB-2; (2) tumor suppressor genes such as p53, p16, DPC4/SMAD4, and DCC; and (3) growth factors such as EGF, FGF, HGF, PDGF, VEGF, TGF-beta. Genetic alterations of K-ras and p53 are common in human pancreatic cancer, but the occurrence of pancreatic cancer is a multi-step phenomenon in which the accumulation of genetic changes is extremely important.
Epidermal Growth Factor ; genetics ; Fibroblast Growth Factors ; genetics ; Genes, Tumor Suppressor ; Genes, myc ; genetics ; Genes, p16 ; Genes, p53 ; genetics ; Genes, ras ; genetics ; Growth Substances ; genetics ; metabolism ; Humans ; Oncogenes ; genetics ; Pancreatic Neoplasms ; genetics

To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed. The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol. The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences. The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage. It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.
Cell Division ; drug effects ; Gene Expression ; Glutathione Transferase ; genetics ; Growth Substances ; genetics ; isolation & purification ; pharmacology ; Humans ; Peptides ; genetics ; isolation & purification ; pharmacology ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Tumor Cells, Cultured

Hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1beta subunit and one of three subunits (HIF-1alpha, HIF-2alpha or HIF-3alpha). The stability and activity of HIF-1alpha are regulated by various post-translational modifications, hydroxylation, acetylation, and phosphorylation. Therefore, HIF-1alpha interacts with several protein factors including PHD, pVHL, ARD-1, and p300/CBP. Under normoxia, the HIF-1alpha subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)- mediated ubiquitin-proteasome pathway. The association of pVHL and HIF-1alpha under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, in the hypoxia condition, HIF-1alpha subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Eventually, HIF-1 acts as a master regulator of numerous hypoxia-inducible genes under hypoxic conditions. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation/survival, and glucose/iron metabolism. Moreover, it was reported that the activation of HIF-1alpha is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1a itself or HIF-1alpha interacting proteins inhibit tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. This review summarizes the molecular mechanism of HIF-1a stability, the biological functions of HIF-1 and its potential applications of cancer therapies.
Alternative Splicing ; Gene Expression Regulation ; Gene Therapy ; Growth Substances/metabolism ; Humans ; Protein Isoforms/chemistry/genetics/*metabolism ; Protein Subunits/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Transcription Factors/chemistry/genetics/*metabolism ; Transcription, Genetic

The regulation roles of insulin-like growth factor-1 (IGF-1) with basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in human nasal septum chondrocytes monolayer culture and cartilage engineering was investigated in this study. The role of IGF-1 with bFGF and TGFbeta2 was investigated by measuring chondrocyte growth kinetic and collagen genes expression. IGF-1 together with bFGF and TGFbeta2 promote cartilage tissue engineering, increase type II collagen expression and enhance the histological features of engineered cartilage.
Cartilage/*transplantation ; Cell Division/physiology ; Chondrocytes/*cytology ; Collagen Type II/genetics ; Culture Media, Serum-Free ; Gene Expression/physiology ; Growth Substances/*physiology ; Insulin-Like Growth Factor I/*physiology ; Tissue Engineering/*methods

OBJECTIVETo investigate protective effects of hHSS transfection against CCl4 or H2O2.

METHODScDNA coding for hHSS was constructed into eukaryotic vector of pcDNA3.1 and transfected into BEL-7402 hepatoma cells. The expression of hHSS was analyzed with Northern blot.

RESULTSThe growth of the hepatoma cells was remarkably enhanced 24 to 144h after hHSS gene transfection, which suggesting hHSS gene expression could stimulate cells activity. Meantime, incubation of both wild-type and vector-transfected as well as hHSS-transfected cells with CCl4 or H2O2 resulted in severe damage as marked by cell mortality and the rate of apoptosis. However, it appeared that the transfection of hHSS enabled the hepatoma cells to raise obvious resistance against CCl4 and H2O2 injury. Compared the vector cells to the vector-transfected cells, apoptosis ratio were (32.44+/-0.52)% and (25.60+/-0.66)% in which treated with CCl4, while (47.78+/-0.45)% and (37.40+/-0.69)% in which treated with H2O2, t value is 16.82 and 25.20, P<0.01. MAPK phosphorylation was also activated after HSS transfected.

CONCLUSIONThe function of hHSS gene expression could be related to proliferation of cell and protection against free radical damage.

Apoptosis ; drug effects ; Carbon Tetrachloride ; toxicity ; Cytoprotection ; Free Radicals ; Growth Substances ; genetics ; physiology ; Humans ; Hydrogen Peroxide ; toxicity ; Liver Neoplasms ; pathology ; Mitogen-Activated Protein Kinases ; metabolism ; Peptides ; genetics ; physiology ; Phosphorylation ; RNA, Messenger ; analysis ; Transfection

AIMTo investigate the protective effects of shark hepatic stimulator substance (sHSS) against acute hepatic injury induced by acetaminophen (AAP) in mice.

METHODSAcute hepatic injury model of Balb/c mice was induced by a single intraperitoneal injection of AAP (200 mg.kg-1, i.p.). Serum ALT and AST activities were analyzed. The changes of microstructure and ultrastructure of hepatocyte were observed under optical and electronic microscope. The hepatocyte apoptosis was analyzed by flow cytometer and the expression level of Fas mRNA was determined by RT-PCR.

RESULTSThe activities of serum ALT and AST were significantly decreased and both necrosis and inflammatory infiltration were improved in the mice treated with sHSS 3.0 and 1.5 mg.kg-1. sHSS (3.0 mg.kg-1) prevented the ultrastructural changes of hepatocytes caused by AAP, decreased the percentage of apoptotic cells, and downregulated the expression level of Fas mRNA.

CONCLUSIONsHSS protected hepatocytes from AAP-induced injury, which might be associated with its protection of the mitochondria and inhibition of apoptosis and expression of Fas mRNA in hepatocytes.

Acetaminophen ; Animals ; Apoptosis ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; pathology ; Female ; Growth Substances ; isolation & purification ; pharmacology ; Mice ; Mice, Inbred BALB C ; Peptides ; isolation & purification ; pharmacology ; Protective Agents ; pharmacology ; RNA, Messenger ; genetics ; Random Allocation ; Sharks ; fas Receptor ; biosynthesis ; genetics

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.
Biological Markers/metabolism ; Cadherins/genetics ; CpG Islands ; *DNA Methylation ; Female ; *Gastric Mucosa/pathology/physiology ; Gene Expression Regulation, Neoplastic ; Growth Substances/genetics ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; PPAR gamma/genetics ; Peptides/genetics ; *Stomach Neoplasms/genetics/pathology ; *Stomach Ulcer/genetics/pathology ; Tumor Suppressor Proteins/genetics ; Wound Healing/*genetics

Hypoxia-inducible factor-1 (HIF-1) is composed of HIF-1alpha and HIF-1beta, and is a master regulator of oxygen homeostasis, playing critical roles in physiological and pathological processes. Normally, the formation and transcriptional activity of HIF-1 depend on the amount of HIF-1alpha, and the expression of HIF-1alpha is tightly controlled by the cellular oxygen tension. Recent progress in the study of its regulation mechanism provided clues as to how HIF-1alpha is regulated by oxygen. It appears that HIF-1alpha is not regulated only by the oxygen tension, but also by various other stimuli, such as transition metals, nitric oxide, reactive oxygen species, growth factors, and mechanical stresses. In this review, we summarize the oxygen-dependent and -independent regulation of HIF-1alpha, and the respective physiological and pathological meanings.
Animals ; Growth Substances/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Molecular Structure ; Nitric Oxide/metabolism ; Oxygen/*metabolism ; Reactive Oxygen Species/metabolism ; Stress, Mechanical ; Transcription Factors/chemistry/genetics/*metabolism ; Transition Elements/metabolism

This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.
Animal ; Cell Division/drug effects/genetics ; Cell Line/cytology/physiology ; Gene Expression/drug effects/immunology ; Graves' Disease/immunology ; Growth Substances/genetics/*pharmacology ; Immunoglobulin G/pharmacology ; Insulin-Like Growth Factor I/pharmacology ; Myxedema/immunology ; Proto-Oncogene Proteins c-fos/*genetics ; Proto-Oncogene Proteins c-myc/*genetics ; RNA/analysis ; Rats ; Rats, Inbred F344 ; Support, Non-U.S. Gov't ; Thyroid Gland/cytology ; Thyrotropin/pharmacology ; Time Factors

AIMTo investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging.

METHODSTwelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%).

RESULTSOur results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats.

CONCLUSIONBesides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.

Aging ; physiology ; Animals ; Antioxidants ; metabolism ; Aromatase ; biosynthesis ; genetics ; Caloric Restriction ; Estradiol ; metabolism ; pharmacology ; Estrogens ; pharmacology ; Lipid Peroxidation ; drug effects ; Male ; Oxidative Stress ; drug effects ; Peganum ; chemistry ; Plant Extracts ; pharmacology ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Receptors, Estrogen ; biosynthesis ; genetics ; Testis ; drug effects ; enzymology ; growth & development ; Testosterone ; metabolism ; Thiobarbituric Acid Reactive Substances ; metabolism