THE EFFECT OF db-cAMP ON THE GENE EXPRESSION OF CALMODULIN AND CYTOSKELETON IN THE TRANSFORMED CELLS

We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 cells. After treatment with 1 mM db-cAMP for 1 hour and 2 hours, there was an early and repldly reduced in gene expression of Calmodulin and c-fos respectively. After db-cAMP treatment for 4 -5 days, the number of capping cells of ConA binding decreased significantly and the cell surface microvllll decreased as well. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed In G1 phase, we have found that the db- cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, mlcrofilaments and fibronectln were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the Inhibition of proliferation, alteration, of phenotype and reco- very of cytoskeleton is transformed cells after treatment with db-cAMP are related to the Inhibition of gene expression of Calmodulin.

DIFFERENCES IN EXPRESSION AND REGULATION BETWEEN TRANSFORMED CELLS OF THE HUMAN GASTRIC CARCINOMA ONCOGENE Ha-ras AND THE UNTRANSFORMED PARENT CELLS

An Ha-ras oncogene was isolated from a cell line of gastric carcinoma called BGE-823 in order to elucidate genetic control and the influence of DNA sequences. The oncogene was cloned and identified as a single nucleotide substitution of thymine for guanine in the 12th codon through the sequencing of its first axon. We compared the differences of expression and regulation between the transformed Ha-ras cells and untransformed parent cells. Data indicated that the expression of Ha-ras in the transformed cells was five-fold higher than in the untransformed cells and that the Ha-ras gene in the former was hypersensitive toward DNase I. In addition, a nuclear protein of 35 kilodaltons bound strongly to the 2.5 Kb fragment located upstream of the 6.6 Kb Ha-ras gene and contained a CC rich region. These results suggest that there might be another mechanism of activation for the ras gene besides point mutation.

VARIATION OF ADHESION CAPABILITY OF K-ras TRANSFORMED MALIGNANT CELLS AND CLINICAL IMPLICATIONS

Objective： To investigate the mechanism of carcinogenesis, invasion and metastasis. Methods： The expressions of adhesive molecule and adhesive structure in v-k-ras transformed normal rat kidney cells （KNRK） were detected with a variety of molecular biological techniques, including cell culture, immunofluorescence labeling, electron microscopy, polyacrylamide gel electrophoresis, and protein blotting, and compared with normal rat kidney （NRK） cells. Results： The significantly shortened doubling time, remarkably active proliferation ability in soft agar, and invasive growth in the abdomen of nude rat, demonstrated the malignant biological behaviors of KNRK cells. In KNRK cells, the adhesive molecules, P-cadherin, α and β catenin, actin, and adhesive structures, the adhesive junction and gap junction, were all abnormally expressed. And cell aggregation was significantly decreased. The aggregation ability disappeared at 20℃, and became active with a suitable amount of calcium solution. Conclusion： Following the transfection of virus K-ras gene, normal cells were transformed into malignant cells. In early stage of cancer, the variation of adhesive ability may be one of the vital factors underlying tumorigenesis, invasion and metastasis.

ELECTRON MICROSCOPIC OBSERVATION OF SIMIAN SARCOMA VIRUS TRANSFORMED NIH 3T3 CELLS IN VITRO

For electronic microscopic observation, we found SSV-transformed NIH 3T3 cells were different from non-transformed cells. In SSV-transformed NIH 3T3 cells nuclei cytoplasma ratio was increased and in cytoplasma the ribosomes (polyribosomes were attached to the swollen rough endoplasmic reticulum. It was likely that ribosomes were lined together functionally and structionally to produce specific protein (PDGF-like protein).

Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Genotoxic and Nongenotoxic Effects of Glycidyl Methacrylate on Human Lung Fibroblast Cells

Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

NEOPLASTIC TRANSFORMATION OF HUMAN EMBRYONIC NASOPHARYNGEAL EPITHELIAL CELLS INDUCED BY N,N'-DINITROSOPIPERAZINE (DNP) IN VITRO

This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.

EDIL3 depletion suppress epithelial-mesenchymal transition of lens epithelial cells via transforming growth factor β pathway

AIM： To study the effect of discoidin I-like domaincontaining protein 3（EDIL3） depletion on the proliferation and epithelial-mesenchymal transition（EMT） in human lens epithelial cells（LECs）. METHODS： RNA interference was used to inhibit the expression of EDIL3 in human LECs in vitro. The morphology of cells was observed using an inverted microscope. Cell proliferation was assessed using Ed U kit. Cell migration was investigated using Transwell chamber and EMT of LECs was assessed using confocal microscope and Western blotting. The transforming growth factor β（TGFβ） pathway was investigated using Western blotting. RESULTS： The data showed that silencing EDIL3 expression changed LECs morphology and suppressed LECs proliferation（P〈0.05） and migration（P〈0.01）. Furthermore, the result of Western blotting showed that EDIL3 depletion reduced the expression of α-smooth muscle actin（α-SMA）（P〈0.001） and vimentin（P〈0.01）, while increased the expression of E-cadherin（P〈0.001）. EDIL3 depletion could suppress the phosphorylation of Smad2（P〈0.01） and Smad3（P〈0.01） and the activation of exracellular signal regulated kinase（ERK）（P〈0.05）. CONCLUSION： The findings indicate that EDIL3 might participate in the proliferation and EMT in LECs via TGFβ pathway and may be a potential therapeutic target for the treatment of posterior capsule opacification.

PI3 kinase isoform p110δis more important than p110αin KIT signaling in hematopoietic cells

PI3 kinases are important for KIT signaling and KIT mutants mediated cell transformation.In order to know the difference of PI3 kinase isoforms p110αand p110δin the signaling of wild-type KIT and the often occurred KIT mutation D816V in hematopoietic malignancy mastocytosis,the predominant PI3 kinase isoform p110δin hematopoietic tissues was knocked out in hematopoietic cells.We found that loss of p110δexpression dramatically inhibits PI3 kinase activation mediated by both wild-type KIT and KIT/D816V.By over expression of p110αin p110δknock out cells,wild-type KIT mediated PI3 kinase activation was not changed while over expression of p110δincreased PI3 kinase activation.Similarly,in KIT/D816V expressing cells without p110δexpression,over expression of p110δbut not p110αrestored PI3 kinase activation.In agreement with the signaling results,cell proliferation,cell survival and cell cycle assay further showed that over expression of p110δbut not p110αin p110δknock out cells increases both wild-type KIT and KIT/D816V mediated cell survival and proliferation.These results suggested that p110δplays a more important role than p110αin KIT signaling and KIT mutant mediated cell transformation in hematopoietic cells.

FURTHER STUDIES ON THE MALIGNANT TRANSFORMATION OF SYRIAN GOLDEN HAMSTER EMBRYO CELLS IN VITR0 BY THORIUM DIOXIDE AND RARE EARTH IRON MINERAL DUSTS

Malignant transformation of hamsterembryo cells was induced in vitro by rareearth iron mineral dusts(MP),naturalthorium(Th02) and MP plus Th02.Dusts of MP,MP plus Th02 or Th02 were added into themedium with the final concentration of 17.0,

Inhibiting Mechanism of Baculovirus p35 Gene to Apoptosis

We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35.

Suppression Effect of Superoxide Dismutase (SOD)-Like Activity Protein Partially Purified from Raphnus sativus Leaves against Liver Metastasis in Mice Intraperitoneally Infected with Ehrlich Ascites Carcinoma Cell

Liver metastasis is a normal result in Ehrlich Ascites Carcinoma Cell (EAC) infected mice. A partially purified protein with Superoxide Dismutase (SOD)-like activity extracted from leaves of Raphnus sativus was tested for its ability to down regulate liver metastasis of Ehrlich Ascites Carcinoma Cell (EAC) intraperitoneally implanted in albino mice. Sixty albino mice were divided equally into 4 groups. Group I mice (G1 control) were treated with physiologic saline solution for 7 days. Group 2 mice (G2) were injected with the partially purified protein. Group 3 mice (G3) were intraperitoneally inoculated with EAC cells and treated with the partially purified protein. Group 4 mice (G4) were inoculated EAC cells and treated with physiologic saline solution. The results showed a disturbance in liver functions. This disturbance was observed as a reduction in the values of serum albumin, hemoglobin, total counts of erythrocytes and platelets, total lipids in liver tissues, erythrocytes SOD activity, serum glucose, reduced glutathione (GSH) and the trace elements (Fe, Mn, Zn, and Cu) contents in liver and spleen tissues as well as the elevation of serum transaminases activities, the levels of malondialdehyde in blood, DNA and RNA of liver tissues in the infected mice (G4). On the other hand, in G3, after treatment with the partial purified protein all these parameters restored their normal values and became near from the normal group (GI). Moreover, the protein showed no toxicity towards the mice as shown in G2. It could be concluded that a partially purified protein with Superoxide Dismutase (SOD)-like activity extracted from leaves of Raphnus sativus as a nature source has a promised future in controlling liver metastasis.

Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (Msp I/Hpa II) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as verified by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

Role of Deoxyribonucleoside Triphosphates(dNTPs)in Cell Transformation

Deoxyribenucleoside triphosphate (dNTP) pools were measured in normal BALB/c3T3 cells, transformation-treated cells and transformed cells with reverse-phase HPLC. The fluctuation of dNTP pools was similar after transformation treatment with alkylating mutagen glycidyl methacrylate(GMA) or Nmethyl- N'- nitro N- nitrosoguanidine (MNNG ). However,the gap between deoxyguanosine triphosphate + deoxyadenosine triphosphate (dGTP + dATP) pools and deoxythymidine triphosphate + deoxycytidine triphosphate (dTTP + dCTP) pools was greatly intensified. The measurements also indicated that the dNTP pools in transformed cells were quite different from those in normal cells. The results suggested that dNTP pools may play an important role in cell transformation

Abnormal expression of c-Myc in human bronchial epithelial cells malignantly transformed by anti-BPDE

Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide(anti-BPDE)is a metabolite of benzo[a]pyrene(B[a]P)and acts as a potent mutagen in mammalian systems.However,molecular mechanisms related to anti-BPDE-induced carcinogenesis are poorly understood.Here,we investigated the expression of proto-oncogene c-myc in human bronchial epithelial cells(16HBE-T)transformed by exposure to anti-BPDE.The levels of mRNA and pro-tein of c-Myc were examined in the 16HBE-T and vehicle-treated control cells(16HBE-N)by using different meth-ods respectively,including reverse transcriptase-polymer-ase chain reaction(RT-PCR),quantitative real-time PCR(Q-PCR),western blot and immunocytochemical meth-ods.The level of c-myc mRNA appeared to be signifi-cantly increased in 16HBE-T,as compared with those of the 16HBE-N.Likewise,the expression of c-Myc protein was significantly enhanced as compared with those of the control cells.Moreover,the localization of c-Myc protein shows mainly nuclear staining in 16HBE-T.In conclu-sion,the abnormal expression of c-Myc was present in anti-BPDE malignantly transformed 16HBE cells,which may be involved in the carcinogenesis molecular mech-anism of anti-BPDE.

Synergistic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and N-nitrosodiethylamine on Cell Malignant Transformation

Objective The present paper aims to investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） and N-nitrosodiethylamine （DEN） on tumorigenesis and its potential mechanism. Methods The potentials of TCDD and DEN in separation or in combination to induce malignant transformation were tested in Balb/c 3T3 cells by using a cell transformation assay method. The possible mechanism of observed effects was studied further by adding α-naphthoflavone （α-NF）, a competitive binding agent of TCDD, to the Aryl hydrocarbon receptor （AhR） pathway. The mRNA expressions of Cyp1a1 and Cyp2a5 gene in Balb/c 3T3 cells treated by DEN and TCDD in separation or in combination with or without presence of α-NF were measured with fluorescence quantification RT-PCR technique. Results The cell transformation frequency （TF） was significantly higher in case of induction with TCDD in combination with DEN, as compared to that with either TCDD or DEN alone. These effects were not inhibited via α-NF. The mRNA expression levels of both Cyp1a1 and Cyp2a5 were enhanced by TCDD treatment alone, but this inducible effect was blocked in cells treated by TCDD and DEN in combination. Conclusion TCDD and DEN had a significant synergistic effect on tumorigenesis when they were used in combination. AhR pathway may not be the key mechanism of this synergistic effect. Thus, it is necessary to further test the potential mechanism involved in cancer development.

Sequential occurrence of T790M mutation and small cell lung cancer transformation in EGFR-positive lung adenocarcinoma:A case report

BACKGROUND The emergence of secondary drug resistance when treating epidermal growth factor receptor(EGFR)mutated non-small cell lung cancer(NSCLC)using EGFRtyrosine kinase inhibitors(EGFR-TKIs),seriously affects the therapeutic efficacy and survival of patients.Here,we report a case of advanced NSCLC focusing on the application of multiple biopsy modalities to reveal the development of multiple resistance mechanisms during targeted therapies.CASE SUMMARY A 54-year-old male patient presented with EGFR 19Del-mutated advanced lung adenocarcinoma,and exhibited the development of a T790M mutation during initial TKI treatment.Following 3 mo of Osimertinib treatment,a mixed response was observed.Tissue biopsy of the progressive lesion showed transformation to small cell lung cancer(SCLC)harboring RB1 and TP53 mutations,with loss of the original T790M mutation.A standard chemotherapy regimen with Anlotinib for SCLC was administered.Repeat biopsy revealed adenocarcinoma combined with SCLC after tumor progression.The patient’s overall survival was 24 mo.CONCLUSION Multiple biopsy modalities can reveal the development of multiple resistance mechanisms which help with treatment decision-making.Comprehensive treatment regimens according to the drug resistance mechanism significantly improved the prognosis of such patients.

Alterations in Retinoic Acid Receptors in Non-Small Cell Lung Cancer and Their Clinical Implications

The nuclear retinoic acid receptor may play a critical role in the process of lung carcinogenesis. Alteration or loss of nuclear retinoic acid receptors (RARs) has been associated with progression in premalignant and malignant tissues and it is associated with malignant transformation in human cells. Vitamin A derivates, such as retinoic acid, have emerged as adjuvant to therapy in several types of cancer with favorable effects. Retinoic acid regulates the expression of target genes through the binding and activation of RARs, inhibiting growth proliferation. Diverse studies have evaluated different retinoids alone or in combination with chemotherapy in lung cancer, from which results have been controversial with benefits observed only in the subpopulation with high levels of triglycerides. Additionally, several large randomized trials using retinoids to prevent tobacco-related cancer have failed;due to the latter the use of retinoids in clinical trials remains controversial. However they could reduce the risk of cancer development in non-smokers. There is evidence that retinoids have different effects on lung cancer;still the identification of biomarkers could determinate their benefits as preventive or therapy agents. This review describes the RAR alterations during the development of Non-Small Cell Lung Cancer and sets out the importance of several cancer treatments with retinoid compounds.