Diversity of Genetic Environment of bla(CTX-M) Genes and Antimicrobial Susceptibility in Extended-spectrum β-lactamase producing Escherichia coli Isolated in Korea

Increasing resistance due to the production of extended-spectrum β-lactamase (ESBL) in Escherichia coli is a major problem to public health and CTX-M enzymes have become the most prevalent ESBL worldwide. In this study, resistance profiles of E. coli isolated in Korea and the genetic environments of bla(CTX-M) genes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of CTX-M. Resistance rates of CTX-M-producing E. coli, including β-lactams, fluoroquinolones and aminoglycosides, were significantly higher than that of CTX-M-non-producers (p<0.01). Of 41 tested, 39 (95.1%) isolates of CTX-M-producing E. coli showed resistance transfer by conjugation. All the transconjugants harboured large plasmids of 118~172 megadalton. Insertion sequence ISEcp1B was detected in the upstream of the bla(CTX-M) in 38 (92.7%) isolates with bla(CTX-M). ISEcp1B was disrupted by IS26 in 16 (39.0%) isolates with bla(CTX-M). ISEcp1B carried −35 and −10 promoter components between right inverted repeat (IRR) and the start codon of bla(CTX-M). orf477 or IS903D was observed in the downstream of the bla(CTX-M) in all the isolates with bla(CTX-M-3/15/55) or with bla(CTX-M-14/27), respectively. Sequence similar to IRR of ISEcp1B was located downstream of orf477. Target duplication sequences were detected both upstream of IRL and downstream of IRR. These results showed the involvement of ISEcp1B in the mobilization of the resistance genes. In conclusion, the surrounding DNAs of bla(CTX-M) genes were very diverse, and the spread and the expression of CTX-M may be deeply related with ISEcp1B. These informations will provide important knowledge to control the increase in CTX-M-ESBLs.

Analysis of Integrons and Antimicrobial Resistances of Multidrug Resistant Escherichia coli Isolated in Korea

Antibacterial drugs are one of the most important therapeutic agents of bacterial infections but multidrug resistant Escherichia coli (MDREC) is an increasing problem worldwide. Major resistance mechanism of MDREC is horizontal gene transfer of R plasmids harboring integrons, which the integron integrase (IntI) catalyzes gene cassette insertion and excision through site specific recombination. In this study, resistance profiles of integron harboring E. coli isolated in Korea and the genetic environments of integron gene cassettes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of integron harboring E. coli. Resistance rates of integron harboring E. coli, including β-lactams, aminoglycosides, and fluoroquinolones and MDR frequencies were significantly higher than that of E. coli without integron (p < 0.01). Majority (80%) of integron harboring E. coli showed resistance transfer by conjugation. Most (80%) of E. coli had dfrA17-aadA5 cassette array and PcH1 hybrid promoter; 16.7% of E. coli had dfrA12-orfF-aadA2 cassette array and PcW promoter. The higher prevalence of weak Pc variants among most (96.7%) of integron harboring MDREC suggests that a flexible cassette array is more important than enhanced expression. All the integrons had LexA binding motif suggests that SOS responses control the expression of these integrons. In conclusion, the genetic bases of integrons were diverse, and the spread and the expression of prevalent gene cassette arrays may be deeply related with strengths of Pc promoters in integrons. These informations will provide important knowledge to control the increase of integron harboring MDREC.

Discovery of a New DNA Gyrase A Inhibitor, 4-(1-methyl-6-nitroquinolin-1-ium-4-yl)amino-N-4-(1-methylpyridin-1-ium-4-yl)aminophenylbenzamide

Escherichia coli (E. coli) is a clinically important causative organism that can lead to urinary tract infections. Quinolone antibiotics are among the first-line treatments for urinary tract infections. However, the frequency of resistance to quinolone in E. coli has been increasing. Therefore, new antimicrobial agents that can be used for treatment in lieu of quinolone antibiotics are needed. In this study, thirty-six compounds with higher scores in a virtual screening based on the three-dimensional structure of E. coli DNA gyrase were selected for in vitro antimicrobial activity testing. An in vitro test confirmed the antimicrobial activity of 4-[(1-methyl-6-nitroquinolin-1-ium-4-yl)amino]-N-[4-[(1-methylpyridin-1-ium-4-yl)amino]phenyl]benzamide (ZINC18057104) against E. coli among the 36 compounds. The minimum inhibitory concentration (MIC) of ZINC18057104 against E. coli ATCC® 25922™ was 2 μg/ml, and the MIC₅₀ and MIC₉₀ for the 72 quinolone-resistant E. coli clinical isolates were 4 and 64 μg/ml, respectively. ZINC18057104, which has a quinoline structure which is similar to the quinolone antibiotics, is predicted to exhibit antimicrobial activity in quinolone-resistant E. coli because it has different molecular interactions with the DNA gyrase than that of existing quinolone antibiotics.

Genome-Wide Association Study of Medication Adherence in Chronic Diseases in the Korean Population

Medication adherence is generally defined as the extent of voluntary cooperation of a patient in taking medicine as prescribed. Adherence to long-term treatment with chronic disease is essential for reducing disease comorbidity and mortality. However, medication non-adherence in chronic disease averages 50%. This study was conducted a genome-wide association study to identify the genetic basis of medication adherence. A total of 235 medication non-adherents and 1,067 medication adherents with hypertension or diabetes were used from the Korean Association Resource project data according to the self-reported treatment status of each chronic disease, respectively. We identified four single nucleotide polymorphisms with suggestive genome-wide association. The most significant single nucleotide polymorphism was rs6978712 (chromosome 7, p = 4.87 x 10-7), which is located proximal to the GCC1 gene, which was previously implicated in decision-making capability in drug abusers. Two suggestive single nucleotide polymorphisms were in strong linkage disequilibrium (r2 > 0.8) with rs6978712. Thus, in the aspect of decision-making in adherence behavior, the association between medication adherence and three loci proximal to the GCC1 gene seems worthy of further research. However, to overcome a few limitations in this study, defining the standardized phenotype criteria for self-reported adherence should be performed before replicating association studies.

Microbial Profile of the Stomach: Comparison between Normal Mucosa and Cancer Tissue in the Same Patient

Gastric cancer is the third most common cancer and the third most frequent cause of cancer mortality in Asia. It is predicted that gastric cancer will remain an important cause of death at least during the next half century because of the increasing number of new cases in an aging population. However, little has been revealed about the role of gastric microbes and their reaction to gastric cancer. In this study, we identified differences in the microbial communities between gastric cancer and normal gastric mucosa by comparing the microbiomes of tissues from the same patients. The clustering analysis results showed different bacterial communities between normal gastric mucosa and gastric cancer. A comparison of bacterial communities at the species level revealed that Helicobacter pylori was significantly reduced in cancer tissue compared to that in normal gastric mucosa in the same patient. A comparison at the genus level showed that Propionibacterium spp., Staphylococcus spp., and Corynebacterium spp. had significantly reduced populations in cancer tissue, whereas Clostridium spp. and Prevotella spp. had significantly increased populations in cancer tissue.

Sonication Induces Apoptosis in HL-60 Cells / 계명의대학술지

To investigate a specific mechanism of apoptosis induced by sonication, we applied 20 kHz ultrasound to leukemia cell line HL-60 with different intensities (0-60 W/cm2) and time durations (0-100 sec). In accordance with previous reports, ultrasound treatment in HL-60 cells induced immediate cell death and delayed cell death which are associated with cell lysis and apoptosis, respectively. Delayed cell death of HL-60 was also detected 5 hours after sonication in our experiment. Detection of caspase activation by Western blot and sub-G1 accumulation by flow cytometry confirmed that apoptosis plays a role in delayed cell death induced by sonication in HL-60 cells. In addition, we found that decrease in lysosomes of HL-60 cells after sonication suggesting lysosomal rupture is involved in the mechanism of cell death induced by sonication.

Curcumin inhibits the expression of COX-2 in UVB-irradiated human keratinocytes (HaCaT) by inhibiting activation of AP-1: p38 MAP kinase and JNK as potential upstream targets

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.

Expression of Human beta-defensin 2 mRNA by Lipopolysaccharide in Human Corneal Epithelial Cells

Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.

Expression of IAP Genes During TPA-Induced Differentiation of HL-60 Cells

HL-60 cells (human promyelocytic leukemia cells) differentiate into the monocyte/macrophage like cells that die spontaneously by apoptosis when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). It is known that inhibitors of apoptosis proteins (IAP) bind to and inhibit caspase 3, 7, 9 activity and the induction of apoptosis. In this study, we examined the expression of IAP genes during TPA induced differentiation of HL-60 cells. During the differentiation, HIAP-1, HIAP-2, and XIAP expressions were decreased in protein levels. The pan-caspase inhibitor z-VAD-fmk blocked the decrease of HIAP-1 and HIAP-2, which indicates HIAP-1 and HIAP-2 could be caspase substrates. These findings suggest that the decrease of IAP proteins is related to the induction of apoptosis that is associated with TPA- induced HL-60 cell differentiation.

Expression of Survivin and HIAP-1 in Korean Gastric Cancers

PURPOSE: Dysregulation of apoptosis may attribute to development of cancer by abnormally prolonging cell viability with accumulation of transforming mutations. Survivin and HIAP (Human Inhibitors of Apoptosis)-1 were recently described as apoptosis inhibitors. Their pathogenic roles in gastric cancer are largely unknown. In the present study, we examined the expression of survivin and HIAP-1 in gastric cancer tissues and cell lines in order to elucidate the roles of survivin and HIAP-1 in the process of gastric carcinogenesis. MATENRIALS AND METHODS: Eight gastric cancer cell lines and five gastric cancer tissues were studied. The expression of survivin and HIAP-1 were evaluated by reverse transcription -polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot. RESULTS: Western blot and RT-PCR analysis revealed survivin and HIAP-1 expression in all gastric cancer cell lines. Increased expression of survivin and HIAP-1 were found in all cases of gastric cancer tissues compared to normal tissues by Western blot analysis. In immunohistochemical analysis tumor cells were stained with anti-survivin and anti-HIAP-1 antibodies. Cell cycle dependence of survivin expression was preserved in gastric cancer cell lines. CONCLUSION: The results indicate that increased expression of survivin and HIAP-1 genes may play an important role in gastric cancer.

Expressional Regulation of Cancer Metastasis Related Gene, MMP-9, by Resveratrol

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). Expression of MMPs is regulated by cytokines and signal transduction pathways including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase of MMP-9 expression and activity. These effects of resveratrol were dose-dependent and correlated with the suppression of MMP-9 mRNA expression levels. PMA caused a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection by MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via AP1 and NFkB response elements. Resveratrol inhibited PMA-mediated activation of c-Jun Nterminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the inhibitory activities of resveratrol on MMP-9, JNK, and PKC-delta may have therapeutic potential for controlling growth and invasiveness of tumors.

FKBP-12 Exhibits an Inhibitory Activity on Calcium Oxalate Crystal Growth in Vitro

Urolithiasis and calcium oxalate crystal deposition diseases are still significant medical problems. In the course of nephrocalcin cDNA cloning, we have identified FKBP-12 as an inhibitory molecule of calcium oxalate crystal growth. lambdagt 11 cDNA libraries were constructed from renal carcinoma tissues and screened for nephrocalcin cDNA clones using anti-nephrocalcin antibody as a probe. Clones expressing recombinant proteins, which appeared to be antigenically cross-reactive to nephrocalcin, were isolated and their DNA sequences and inhibitory activities on the calcium oxalate crystal growth were determined. One of the clone lambdagt 11 #31-1 had a partial fragment (80 bp) of FKBP-12 cDNA as an insert. Therefore, a full-length FKBP-12 cDNA was PCR-cloned from the lambdagt 11 renal carcinoma cDNA library and was subcloned into an expression vector. The resultant recombinant FKBP-12 exhibited an inhibitory activity on the calcium oxalate crystal growth (Kd=10(-7) M). Physiological effect of the extracellular FKBP-12 was investigated in terms of macrophage activation and proinflammatory cytokine gene induction. Extracellular FKBP-12 failed to activate macrophages even at high concentrations. FKBP-12 seems an anti-stone molecule for the oxalate crystal deposition disease and recurrent stone diseases.

Expression of Cell Cycle Regulatory and Apoptosis-related Proteins in Etoposide-treated Human Skin Fibroblast / 대한암학회지

PURPOSE: This study was designed to investigate effect of the etoposide on expression of cell cycle regulatory proteins and apoptosis-related proteins in human skin fibroblast (HSF). MATERIALS AND METHODS: HSF cells were treated with etoposide. After treatment, expression patterns of cell cycle regulatory proteins and apoptosis-related proteins were analyzed by using Immunoprecipitation-Western blot method and RT-PCR. RESULTS: Immediately after etoposide treatment, E2F-1 was up- regulated following MDM2 down-regulation. After E2F-1 up-regulation, p53 and p21WAF1 level was markedly increased without or with mRNA up-regulation, respectively. Consistent with these results, cell cycles arrested in mainly G1 phase 24 hr after etoposide treatment. However, HSF cells progressed into apoptosis 72 hr after etoposide treatment. In this process, caspase-3 activation and Bax up-regulation were observed. CONCLUSION: In early response of etoposide treatment, E2F-1 plays an important role in p53 accumulation through MDM2 down- regulation. The increased p53 induce an increase of p21WAF1 level through p21WAF1 mRNA up-regulation. However, after long term treatment of etoposide, HSF cells resulted in apoptotic cell death through caspase-3 activation and Bax up-regulation.

The Effects of Epigallocatechin Gallate on Ultraviolet B Irradiated Cultured Human Skin Fibroblasts / 대한피부과학회지

BACKGROUND: The main polyphenol components in green tee are (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate (EGCG). It is well known that flavonoids such as catechins can be protective against inflammatory and cancer and cardiovascular diseases. These protective effects are largely due to their inhibition of some enzymes and antioxidative activities by scavenging free radicals. Ultraviolet(UV) exposure of the skin, particulary UVB (290-320nm), causes adverse biological effects, including alterations in cutaneous immune cells, photoaging and photocarcinogenesis. Several studies have shown that EGCG afforded protection against UVB-induced inflammatory responses and photocarcinogenesis in murine models. OBJECTIVE AND METHODS: In this study, we investigated the effects of EGCG on UVB irradiated human skin fibroblasts using viability test, thiobarbituric acid assay, propidium iodide(PI) stain, and western blot analyses and RT-PCR. RESULTS: Cell survival curves after UVB irradiation showed dose dependent decrement pattern by trypan blue exclusion assay. Only 42% of dermal fibroblasts survived at 150 mJ/cm2 UVB irradiation. The damage was associated with cell membrane lipid peroxidation, as shown by accumulation malondialdehyde(MDA). By pre-cultivation with EGCG (50nmol), a significant preventive effect was noted on the increase in the absolute number of surviving cells(up to 81.5% of cells survived) and the levels of MDA markedly decreased. Morphological changes associated with apoptotic cell death were easily distinguished by PI stain. Bases on our finding, we investgated the regulation of p53, p21, bax, bcl-2, cyclin D1, E, Cdk2, and PARP proteins by western blot analyses. The expression p53 protein was elevated by following UVB exposure which was inhibited by EGCG treatment. Using RT-PCR, the transcription of p53, fas and jun gene showed similar results which obtained by western blot analyses. CONCLUSION: EGCG, which have newly accepted as a potential UV protection properties, is effective membrane peroxidation inhibitor and prevent apoptotic changes when present in relevant concentration at the site of action beginning and during UVB irradiation. And the protective mechanism of EGCG against UVB-induced cell damage maybe, at least in part, related with p53, fas and jun pathway.

The Levels of MDM2 Protein Are Decreased by a Proteasome-Mediated Proteolysis Prior to Caspase-3-Dependent pRb and PARP Cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.

T Cell Chemokine Inducibility of Lipopolysaccharide, Interleukin (IL)-12/IL-2 and Interferon Gamma in Mouse Brain

BACKGROUND: Interferon gamma (IFN-gamma)-inducible protein 10 (IP-10) and monokine induced by IFN-gamma(Mig) are members of CXC-chemokine family, that are produced from macrophage/monocyte activated by IFN-gamma. These chemokines especially recruit activated T cell into inflammatory site. Here, we studied effect of lipopolysaccharide (LPS), interleukin (IL)-12/IL-2 or IFN-gammaon induction of MIG and IP-10 in mouse brain. METHODS: In order to evaluate Mig and IP-10 gene expression in brain, we injected LPS, IFN-gammaor IL-12/IL-2 into mice intraperitoneally, and measured chemokine message in brain by RT-PCR. RESULTS: In vivo injection of LPS induced Mig and IP-10 gene expression in brain of normal mice and IFN-gammaknockout mouse, however, in vivo injection of IL-12/IL-2 induced Mig and IP-10 gene expression in only the brain of normal mice. IFN-gammainduced chemokine expression in cultured brain cells, but anti-inflammatory drugs did not block IFN-gammaeffects. CONCLUSIONS: Immune stimulating agents, LPS or IL-12/IL-2 or IFN-gamma, can induce T cell chemokine gene expression in brain cells, and these chemokines may play a role in T cell infiltration in various brain diseases. (J Korean Neurol Assoc 19(2):155~162, 2001)

T Cell Chemokine Inducibility of Lipopolysaccharide, Interleukin (IL)-12/IL-2 and Interferon Gamma in Mouse Brain

BACKGROUND: Interferon gamma (IFN-gamma)-inducible protein 10 (IP-10) and monokine induced by IFN-gamma(Mig) are members of CXC-chemokine family, that are produced from macrophage/monocyte activated by IFN-gamma. These chemokines especially recruit activated T cell into inflammatory site. Here, we studied effect of lipopolysaccharide (LPS), interleukin (IL)-12/IL-2 or IFN-gammaon induction of MIG and IP-10 in mouse brain. METHODS: In order to evaluate Mig and IP-10 gene expression in brain, we injected LPS, IFN-gammaor IL-12/IL-2 into mice intraperitoneally, and measured chemokine message in brain by RT-PCR. RESULTS: In vivo injection of LPS induced Mig and IP-10 gene expression in brain of normal mice and IFN-gammaknockout mouse, however, in vivo injection of IL-12/IL-2 induced Mig and IP-10 gene expression in only the brain of normal mice. IFN-gammainduced chemokine expression in cultured brain cells, but anti-inflammatory drugs did not block IFN-gammaeffects. CONCLUSIONS: Immune stimulating agents, LPS or IL-12/IL-2 or IFN-gamma, can induce T cell chemokine gene expression in brain cells, and these chemokines may play a role in T cell infiltration in various brain diseases. (J Korean Neurol Assoc 19(2):155~162, 2001)

Alterations of p15INK4B, p16INK4A and Methylthioadenosine Phosphorylase Gene in Korean Hepatdegrees Cellular Carcinoma / 대한암학회지

PURPOSE: We analyzed the gene status of p16INK4A, p15INK4B and MTAP (methylthio adenosine phophorylase) in Korean hepatdegrees Cellular carcinoma (HCC) to investigate whether the inactivation of these genes participated in hepatdegrees Carcinogenesis, and evaluated MTAP-targeted chemotherapy in MTAP-deficient cell lines. MATERIAL AND METHODS: We examined eleven primary HCC and 8 SNU cell lines using PCR, Southern blot analysis, PCR-SSCP, DNA sequencing, methylation-specific PCR, Western blot analysis, MTT assay, and crystal violet staining. RESULTS: Mutations or deletion of the p16INK4A, 15INK4B, and MTAP genes were rare, but methylation of the p16INK4A promoter region was common in HCC. The base alterations of 3' untranslated region of p16INK4A exon 3 were also detected in 3 samples. In SNU cells, p16INK4A was not detectable, when treated with demethylating agent, high levels of re-expressed p16INK4A protein were detected. In MTAP-targeted chemotherapy experiment, methylthioadeno sine (MTA) was able to rescue MTAP positive cell lines but not MTAP negative cell lines from growth inhibition by depletion of methionine and MTX treatment. CONCLUSION: These results suggest that de novo methylation of the p16INK4A promoter region seems to play an important role in the pathogenesis of HCC. And treatment of MTX, combined with methionine depletion in the presence of MTA, may be a high selective treatment for MTAP negative HCC.

Expressions of Cell Cycle Control Genes in Human Uterine Cervical Cancer Cells / 대한암학회지

PURPOSE: Recently, many aspects of biological functions of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors and Rb gene have been reported, and the cell cycle control genes are considered to act important roles in tumorigenesis. In this study, the expression patterns of major cell cycle control genes (cyclin A, B, C, Dl, E, E2F, p16INK4a, p21WAF1 and Rb) in various human cervical cancer cells were analysed to elucidate the impacts of the cell cycle control genes on the carcinogenesis of human cervical cancer. MATERIALS AND METHODS: The expression patterns of major cell cycle control genes in HT-3, C33-A, HeLa, C4-II, SiHa and CaSki human uterine cervical cancer cells were analysed by using western blot and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In most of the cervical cancer cells tested, the overexpressions of cyclin A, E, E2F and markedly decreased expression of Rb tumor suppressor proteins were observed. By comparing RNA and protein expressions in each cancer cells, the mechanisms of increased expressions of cyclin A, E and decreased expression of Rb were elucidated as post-translational controls. CONCLUSION: The cervical carcinogenesis caused by the altered expressions of the major cell cycle control genes can be hypothesized as follows: overexpressions of cyclin E and A cause acceleration of Rb phosphorylations and E2F overexpression; increased E2F function accelerates G1/S transition of the cells; compensatory increase of p16 expression cannot stop the cells in Gl phase because Rb expression is severely decreased; consequently, loss of Rb function, 61 shortening, inappropriate cell division and decreased function of the maintenance of genomic stability occur. In addition to these alterations, loss of p53 functions further accelerate instability of genome and decrease the sus- ceptability to cell death. Furthermore, overexpression of Bc12 protects these abnormal cells from apoptosis. All these derangements of cell cycle control should contribute to the human cervical carcinogenesis.

Construction of MAGE - 3 Expressing Plasmid for Development of DNA Vaccine Encoding MAGE - 3 Cancer Antigen / 대한암학회지

PURPOSE: The spectrum of melanoma antigen gene (MAGE)-expressing tumor is very wide and the gene of MAGE express antigens that are targets for specific recognition by cytotoxic T lymphocytes derived from tumor-bearing patients. All of these characteristics represent MAGE as tumor vaccine can be useful for cancer prevention or treatment. Here, we detected MAGE-3 gene expression in cancer cell lines and evaluated recombinant MAGE-3 protein producibility of MAGE plasmid to develope MAGE DNA vaccine. MATERIALS AND METHODS: MAGE-3 gene expression of cancer cell lines was evaluated by reverse transcription-polymerase chanin reaction (RT-PCR). Two kinds of MAGE-3 expressing plasmids were constructed and their MAGE-3 protein producibility was evaluated by immunohistochemistry and immunoblotting using monoclonal anti-MAGE-3 antibody. RESULTS: Among 13 cell lines, SNU484, AMC-HN-3, AMC-HN-4, AMC-HN-7, HeLa, NCI H1703 and HT29 expressed MAGE-3 mRNA. In order to make MAGE plasmid, cDNA that showed 100% DNA homology with MAGE-3 gene was cloned into pcDNA 3 plasmid and pSecTag plasmid. Intracytoplasmic and secretory recombinant MAGE-3 was produced by MAGE-3 containing pcDNA 3 plasmid and pSecTag plasmid, respectively. CONCLUSION: In this study, we showed high expression frequency of MAGE-3 in cancer cell line, and established two kinds of plasmid that produce recombinant MAGE-3 in cell lines. We expect these plasmids will be used in cancer treatment or MAGE-3 function study in future.