The effect of four alkylating agents (MMS, EMs, DMN, DEM), under various con centrations on mouse bone marrow erythrocytes, were studied by means of the micronucleus test. The results obtained were as follows: 1) The lethal doses on mice were MMS = 130 mg/kg/bw, EMS = 300 mg/kg/bw, DMN = 50 mg/kg/bw and DEN = 70 mg/kg/bw. 2) Micronuclei were easily seen and in different controls the micronulei were found a little over 0.1%. 3) The dose-effect relationship was obtained. In the MMS and EMS treated groups, incidences of micronulei were 0.45 to 2.56% and 0.4 to 2.1% respectively. 4) In the DMN and DEN treated groups, incidences varied between 0.15 to 0.90 % and 0.2 to 1.02% respectively. 5) Four alkylating agents were compared and discussed with respect to micro nucleus production from chromosomal aberrations.
Animal ; Bone Marrow/ultrastructure ; Carcinogens/pharmacology* ; Cell Nucleus/drug effects* ; Chromosome Aberrations* ; Erythrocytes/ultrastructure ; Female ; Mesylates/pharmacology* ; Mice ; Mutagens/pharmacology ; Nitrosamines/pharmacology*

OBJECTIVETo clone differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene (BPDE).

METHODThe malignant transformation of human bronchial epithelial cell line 16HBE induced by BPDE in vitro was used as a model for comparing gene expression between the transformed cells and controls. cDNA representational difference analysis (cDNA-RDA) was performed to isolate differentially expressed cDNA fragment in transformed cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA were sequenced and compared with data in GenBank by BLASTN.

RESULTSFive cDNA sequences were found to be novel ones and were registered in dbest database, which assigned accession numbers in GenBank are BG354691, BG354692, BG354693, BG354694 and BG354695, respectively. Eight of the remaining cDNA sequences showed sequence homology to those previously reported such as ribosomal protein S23, MLN137, ACTN4, transforming growth factor and G protein gene.

CONCLUSIONSThese 13 genes may be involved in BPDE-induced malignant transformation, but their biological characteristics and functions are left to further studies.

Benzopyrenes ; metabolism ; pharmacology ; Carcinogens ; pharmacology ; Cell Transformation, Neoplastic ; chemically induced ; genetics ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; analysis ; drug effects ; DNA, Neoplasm ; analysis ; Gene Expression ; drug effects ; Humans

OBJECTIVETo establish an in vitro heterogeneous expression model of human CYP2E1 (hCYP2E1) cDNA and investigate the effect of the chemical carcinogenic N, N'-dinitrosopiperazine (DNP) on the expression of CYP2E1.

METHODSExogenous hCYP2E1 was introduced into the mouse derived NIH3T3 cells using the lipofectamine transfection technique. Integration of exogenous hCYP2E1 gene was identified by PCR and Southern blot. After treatment with various concentration of ethanol and DNP on the transfected NIH3T3 cell cultures, RT-PCR and Western blot was applied to detect the expression level of CYP2E1.

RESULTSTwo cell clones with integration and stable expression of exogenous hCYP2E1 were obtained and designated as NIH3T3-2E1-A4 and NIH3T3-2E1-A8 respectively. The expression of both hCYP2E1 mRNA and protein products was promoted after either ethanol or DNP treatment.

CONCLUSIONThe results suggested that the promoted expression of hCYP2E1 induced by DNP and /or ethanol is due to enhanced transcription. The mechanism of DNP carcinogenes is might be related to this in situ activated metabolism by CYP2E1.

3T3 Cells ; Animals ; Carcinogens ; toxicity ; Cytochrome P-450 CYP2E1 ; genetics ; Ethanol ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Mice ; Nitrosamines ; toxicity ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Aflatoxin B1 (AFB1), a fungal toxin produced by Aspergillus flavus, is known to be a possible hepatocarcinogen. But the molecular biologic changes which may occur following exposure to AFB1 are not known and thus the carcinogenesis is not yet understood. This study was performed to examine the expressions of c-myc, c-fos and TGF-alpha genes and to investigate the possible role of those molecular biologic changes in hepatic regeneration and in the development of hepatocellular carcinoma (HCC). Sprague-Dawley rats were divided into 3 groups: Carbon tetrachloride (CCl4) only was administered to group I, AFB1 only was administered to group II and a combination of AFB1 and CCl4 was administered to group III. The animals were sacrificed at 0.5, 1, 2, 6, 12, 24, 48, and 72 hours after treatment. In addition to the examination of the hematoxylin-eosin stained sections, hepatic regeneration and apoptosis were analyzed quantitatively by bromodeoxyuridine (BrdU)-anti-BrdU immunohistochemistry and TUNEL assay utilizing apoptosis kit, respectively. The hepatic expressions of c-myc, c-fos and transforming growth factor-alpha (TGF-alpha) were examined by immunohistochemistry and studied by Western blot. The number of BrdU labelled cells and the degree of necrosis/apoptosis were comparable among the different groups. Livers of the group II rats showed nearly normal histology without regeneration and necrosis/apoptosis. In groups I and III, the number of BrdU- labelled cells showed an increase at 48 hours after treatment, and the increment was significantly higher in group I than in group III. Most BrdU-labelled cells were mature hepatocytes in group I, whereas in group III they appeared to be less mature. In group I, apoptosis showed an increase at around 24 hours, but appeared in group III as early as 12 hours after treatment and persisted through 48 hours. The expression of c-myc and c-fos were also different between the experimental groups. The expression intensity of c-myc in group I was highest at 1 hour and decreased thereafter. In groups II and III, the expressions were much more intense than in group I, except at 1 hour, and the increased intensity persisted throughout the experiment. Group II in particular showed a peak intensity at 30 minutes and at 6 hours after treatment. In group I, c-fos was strongly expressed only at 24 hours, but in group III, there was progressively increased expression with peak intensity at 24 hours. TGF-alpha was expressed in similar intensities in all groups throughout the experiment. These results suggest that AFB1 may evoke an intense and protracted expression of c-myc, provocating the CCl4-induced necrosis of hepatocytes, and a prolonged expression of c-fos, including persistent signals for regeneration which in turn may activate the replication of immature cells. These findings will aid further investigation of molecular biologic and histologic characteristics of the hepatotoxic and hepatocarcinogenic mechanism of AFB1 in rats. And these results in rats, together with clinico-epidemiologic and molecular biologic investigations in humans and other animals, suggest that AFB1 may supply hepatocarcinogenic background in early exposure time in AFB1-contaminated areas of China and Korea.
Aflatoxin B1/pharmacology* ; Animal ; Carbon Tetrachloride ; Carcinogens/pharmacology* ; Gene Expression/drug effects* ; Genes, Immediate-Early/drug effects* ; Liver Diseases/metabolism* ; Liver Diseases/genetics* ; Liver Diseases/chemically induced ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor alpha/metabolism*

To evaluate the induction of preneoplastic hepatic foci in relation to natural killer cell (NK) activity, we sequentially analyzed glutathione S-transferase placental form positive (GST-P+) hepatocytes and NK activity during diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis in Sprague-Dawley rats. Previous studies have shown that NK activity can modulate the carcinogenic process induced by chemical carcinogens. Newborn females were initially given a single intraperitoneal injection of 15 mg DEN/kg and three weeks later, they were treated with 500 ppm phenobarbital (PB). From week 3, PB was administered in drinking water for 9 weeks. Interim and terminal sacrifices were performed at weeks 12, 15 and 30. GST-P+ hepatocytes increased with age in DEN-treated rats, especially in the population of more than two GST-P+ hepatocytes. The NK activity of DEN-treated rats did not significantly differ from that of control rats until week 12, but it progressively decreased from week 15 to 30. These results indicate that changes of NK activity inversely correlated with the induction of preneoplastic hepatic foci. This strong correlation of decreased NK activity with enhanced induction of GST-P+ foci suggests that NK activity is important in the early progression of hepatocarcinogenesis in rats.
Animal ; Body Weight ; Carcinogens/pharmacology* ; Diethylnitrosamine/pharmacology* ; Female ; Glutathione Transferase/metabolism* ; Killer Cells, Natural/immunology* ; Liver/enzymology* ; Liver/cytology ; Liver Neoplasms/physiopathology* ; Organ Weight ; Placenta ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells).

METHODSThe suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank.

RESULTSEight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database.

CONCLUSIONThe SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ; pharmacology ; toxicity ; Carcinogens ; pharmacology ; toxicity ; Carcinoma ; genetics ; metabolism ; Cell Line ; Cell Line, Transformed ; Cell Transformation, Neoplastic ; chemically induced ; Gene Expression Regulation, Neoplastic ; Humans ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; RNA, Messenger ; metabolism

The inhibitory effects of ginseng on the development of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in the colon were investigated in rats. Male, 6-week-old rats were injected with DMH once a week for 4 weeks. Rats in Groups 1 and 2 were fed diets containing red and white ginseng, rerspectively, at a dose of 1% for 5 weeks, starting one week before the first treatment of DMH. Animals in Groups 3 and 4 received red or white ginseng for 8 weeks starting after DMH treatment. Group 5 served as a carcinogen control group. Numbers of ACF with at least four crypts were significantly reduced in the colon of Group 2 treated with red ginseng combined with DMH. Moreover, rats were injected with DMH 4 times at one-week intervals. They were also fed diets containing 1% red or white ginseng or the control diet throughout 30 days of the experiment. Treatment with red ginseng resulted in a significant decrease of 5- bromo-2'-deoxyuridine labeling indices in colonic crypts comprising ACF. These findings suggest that dietary administration of red ginseng in combination with DMH suppresses colon carcinogenesis in rats, and the inhibition may be associated, in part, with inhibition of cell proliferation, acting on ACF in the colonic mucosa.
1,2-Dimethylhydrazine/adverse effects ; Animal ; Anticarcinogenic Agents/*pharmacology ; Carcinogenicity Tests ; Carcinogens/adverse effects ; Colonic Neoplasms/pathology/*prevention & control ; Male ; *Panax ; Plant Roots ; Precancerous Conditions/pathology/*prevention & control ; Rats ; Rats, Inbred F344

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type-Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer.
Proto-Oncogene Proteins c-met/*metabolism/*physiology ; Protein Isoforms/metabolism/physiology ; NIH 3T3 Cells ; Mutant Proteins/metabolism/physiology ; Mice, Nude ; Mice ; Hepatocyte Growth Factor/pharmacology ; Female ; Down-Regulation ; Carcinogens/*metabolism ; Carcinogenicity Tests ; Animals ; *Alternative Splicing

OBJECTIVETo investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.

METHODSPhorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.

RESULTSPMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.

CONCLUSIONSSphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Apoptosis ; drug effects ; Carcinogens ; administration & dosage ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; HT29 Cells ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; physiology ; Sphingosine ; administration & dosage ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; administration & dosage ; analogs & derivatives ; pharmacology ; Time Factors ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the main proteinases responsible for CD16b shedding under different stimulators.

METHODSHEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.

RESULTSHEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.

CONCLUSIONSBoth ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

ADAM Proteins ; genetics ; metabolism ; physiology ; ADAM10 Protein ; ADAM17 Protein ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; physiology ; Calcium Ionophores ; pharmacology ; Carcinogens ; pharmacology ; Cells, Cultured ; Drug Evaluation, Preclinical ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ionomycin ; pharmacology ; Membrane Proteins ; genetics ; metabolism ; physiology ; Protein Processing, Post-Translational ; drug effects ; Protein Transport ; drug effects ; Proteolysis ; drug effects ; Receptors, IgG ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Transfection