Differential protein expression of etoposide-treated CaSki cervical carcinoma cells / 부인종양

OBJECTIVE: This study was designed to examine the pharmaco-dynamic pattern of proteomic expression in cervical carcinoma cells (CaSki cell line; HPV-16 positive) after in vitro treatment by the etoposide. METHODS: We analyzed proteomic profiling in cervical carcinoma cells after etoposide treatment using two-dimensional gel electrophoresis (2-DE) with MALDI-TOF-MS used for protein identification. Then, we tested the several experimental methods for verification and functional identification, including MTT assay, PI staining, DNA fragmentation assay, FDA, FACS and Western blot analysis. RESULTS: Etoposide inhibited the CaSki cervical cancer cell growth in a dose-dependent manner and the optimal concentration of etoposide is 2micrometer(IC50) in the CaSki cervical cancer cells. The etoposide induced apoptosis, as determined by DNA fragmentation assay, FACS, and Western blot. The etoposide increased the protein expression of Fas (Apo-1/CD95), p53, pRb and caspase-3, but decreased the level of Bcl-2 and caspase-3 precursor and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9). To this end, we analyzed CaSki cancer cells using 2-DE. Eight proteins (XAP-5, HXC-36, serine/threonine protein phosphatase 2B catalytic subunit, G2/mitotic-specific cyclin B1, T-box transcription factor TBX20, diacylglycerol kinase, amiloride-sensitive amine oxidase, HEF-like protein, ras-related protein Rab-20) were down-regulated and nine proteins (RNA 3'-terminal phosphate cyclase-like protein, late endosomal/lysosomal Mp1 interacting protein, glia maturation factor, replication protein A 14 kDa subunit, mago sashi protein homolog, 14 kDa phosphohistidine phosphatase, protein C14 or f48, cyclin-dependent kinase 4 inhibitior A, retinoic acid-binding protein II) were up-regulated in etoposide-treated CaSki cells when compared with non-treated cells. CONCLUSION: Our results clearly indicate that etoposide induced cell death by apoptosis. These findings may provide insights into the mechanisms underlying the apparent anti-tumoral effects of etoposide.

Identification of TACC3 (Transforming Acidic Coiled-Coil 3) as a novel target of paclitaxel-mediated tumor therapy in cervical cancer cells / 부인종양

OBJECTIVE: Paclitaxel is currently used in the treatment of ovarian, breast, gastric, colorectal, lung and recurrent cervical cancer. Initial studies on the mechanism of action of paclitaxel have demonstrated that this drug alters microtubule assembly, by inhibiting microtubule depolymerization and changing microtubule dynamics. Although treatment of various tumor cells with paclitaxel induces apoptosis, but early paclitaxel-targeted proteins is not yet known. We tried to search paclitaxel-targeted proteins and to investigate its functions. METHODS: The effects of paclitaxel on HeLa cervical cancer cell growth were evaluated by cell proliferation assay, DAPI stain, and FACS analysis. We performed proteome analysis including 2-DE and MALDI-TOF-MS in nontreated-and paclitaxel-treated HeLa cells, as a result, we identified TACC3 protein that is down-regulated with paclitaxel treatment. We tried to characterize TACC3 functions through in vitro treatment of paclitaxel or RNAi technique. RESULTS: Paclitaxel- and TACC3 siRNA-treated cells are unable to proceed normally through the cell cycle and are arrested in G2/M phase and reveal apoptotic morphology. TACC3 levels after paclitaxel treatment decreased as a time- and dose- dependent manner both mRNA and protein levels. We confirmed that the role of TACC3 down-regulation for microtubule stabilization was similar to that of paclitaxel. Also, TACC3 is expressed at high levels in various cancer cells and tumor tissues. CONCLUSION: This study is proposed that the TACC3 protein may be participated in microtubule formation as an oncoprotein during mitosis and be regulated by paclitaxel as a novel target.

Transcutaneous DNA vaccination increase the CTL response: Preliminary results for the therapeutic HPV DNA vaccination / 부인종양

OBJECTIVE: Transcutaneous immunization (TCI) is a novel vaccination based on the application onto bare skin. We compared the immune response after TCI with the model DNA (OVALBUMIN) to HPV E7 and various adjuvant with intramuscular injection. We investigated the efficacy of immunization with new construct driven by K6hf promoter and compared with CMV promoter. METHODS: First, we make new construct ligated with OVA to Hair-follicle Specific pK6hf Promoter and evaluated the expression. Mouse skin was transfected with pCMV-OVA, pK6hf-OVA, pCMV-beta gal and pK6hf-beta gal and expression was determined by RT-PCR and X-Gal staining. OVA protein expression was analyzed by Western blot. Second, we immunized C57/ BL6 mice with pCMV-OVA or pK6hf-OVA DNA and cholera toxin (CT) and/or CpG. CTL was measured by ELISPOT assay of the splenocytes from the mmunized mice with the DNA vaccine. RESULTS: The beta-Galactosidase activity by X-Gal staining was detected in the epithelium of the mice skin after pK6hf-beta gal application. The mRNA and protein expression from pK6hf-OVA were evident following transcutaneous methods. Those were weaker than pCMV-OVA. TCI with pCMV-OVA and LipofectAMINE 2000 trigered an speicific CTL and Th2 response. CpG was the adjuvant for CTL after pCMV-OVA. CT and CpG did increase the CTL after pK6hf-OVA. CONCLUSION: Our data demonstrate that TCI of DNA is possible methods of CTL. CpG and CT were useful in the adjuvant for CTL. The pK6hf-OVA can induce specific CTL. This result is of potential relevance for the development of therapeutic HPV- specific DNA vaccines with TCI and pK6hf promoter can be used safely.

The Role of HPV E6 and E7 Oncoproteins in HPV-associated Cervical Carcinogenesis / Journal of the Korean Cancer Association, 대한암학회지

Cervical cancer is one of the leading world causes of cancer morbidity and mortality in woman, with more than 98% related to a human papillomavirus (HPV) infection origin. Infection with specific subtypes of HPV has been strongly implicated in cervical carcinogenesis. The identification and functional verification of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis and the development of cervical cancer-specific markers. The advent of functional genomics and proteomics has provided hope of discovering novel biological markers for use in the screening, early diagnosis, prognostication and prediction of response to therapy. Herein, we review the studies where the profiles of host proteins associated with HPV E6 and E7 oncoproteins in cervical cancer were generated.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.

Proteome Analysis of Differential Protein Expression in Cervical Cancer Cells after Paclitaxel Treatment / Journal of the Korean Cancer Association, 대한암학회지

@#PURPOSE: It is well known that infection with HPV (human papillomavirus) is the main cause of cervical cancer and certain types of HPV are recognized as carcinogens. At present, there is little information regarding the antineoplastic mechanism of paclitaxel against cervical carcinoma cells. We thus tried to analyze differential protein expression and antineoplastic mechanism-related proteins after paclitaxel treatment on cervical cancer cells by using a proteomic analysis and to investigate the mechanism of action. MATERIALS AND METHODS: Using proteomics analysis including 2-DE and MALDI-TOF-MS, we detected the antineoplastic mechanism-related proteins. Then, we performed western blot analysis for apoptosis- and transformation- related proteins to confirm expression patterns derived from proteome analysis after paclitaxel treatment. RESULTS: We identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells using proteomics methods. Paclitaxel treatment elevated main-ly apoptosis, immune response and cell cycle check point- related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Also, in the HPV-associated cervical carcinoma cells, paclitaxel demonstrated anti-proliferative activity through the membrane death receptor-mediated apoptotic pathway and the mitochondrial-mediated pathway. CONCLUSION: Identification and characterization of functionally modulated proteins involved in anti-cancer regulatory events should lead to a better nderstanding of the long-term actions of paclitaxel at the molecular level and will contribute to the future development of novel therapeutic drug treatments based upon current therapies.

Detection of Leptospiral DNA from Field Rodents by PCR

Leptospirosis has been one of important epidemic diseases in Korea since 1984. Wild rodents, mostly Apodemus agrarius, served the important source of infection especially in harvest season in rural areas of Korea. Prevalence of Leptospira spp. infection in field rodents were investigated by detecting leptospiral DNA from rodent kidney. Among 108 rodents collected from various areas in 1998, leptospiral DNA were detected from 7 rodents (6.48%). Among 104 rodents, Apodemus agrarius, captured from Yeonchon and Paju area in 2001 and 2002, leptospiral DNA were detected from 6 rodents (5.76%). No leptospiral DNA was detected from 23 rodonts (Apodemus peninsulae 16, Apodemus agrarius 2 and Eothenomys regulus 5) captured in Odae mountain area in 1998.