Therapeutic advantage of genetically engineered Salmonella typhimurium carrying short hairpin RNA against inhibin alpha subunit in cancer treatment.

Background: In contrast to its well-known endocrine function, the role of inhibin in cancer development and therapeutic response is unclear. Salmonella, particularly less toxic attenuated Salmonella strains, are used to treat cancer in two ways. First, Salmonella accumulate around tumors, penetrate the cell barrier, and replicate inside the tumors. Second, Salmonella can act as a vehicle for delivering anticancer agents or proapoptotic genes to attack tumors. In this study, we aimed to develop a suitable cancer therapeutic strategy by genetically modifying attenuated Salmonella typhimurium to harbor short hairpin RNA (shRNA) expression plasmids targeting alpha subunit of inhibin (sh-INHA). Methods: We analyzed the expression of human INHA in normal and cancer cells and tissues. We developed genetically engineered attenuated S. typhimurium harboring sh-INHA (S. typhimurium/sh-INHA) and assessed its cancer therapeutic effects by using cell culture models and syngeneic mouse tumor models. Results: INHA expression levels were markedly higher in colon cancer and melanoma cells and tissues than in their normal counterparts. Suppression of INHA expression mildly reduced cancer cell survival and induced caspase activation and downregulation of anti-apoptotic Bcl-2 and Bcl-xL expressions. Although the genetically engineered S. typhimurium mildly interfered with the invasion of S. typhimurium into host colon cancer and melanoma cells, S. typhimurium/sh-INHA caused remarkable cytotoxicity in cancer compared with unmodified S. typhimurium or S. typhimurium expressing a control scrambled shRNA (S. typhimurium/sh-Cont). Salmonella typhimurium/sh-INHA-treated mice also showed a significantly inhibited growth of colon cancers and melanomas, with a survival advantage. Conclusion: Our results suggest that tumor-targeted therapy using S. typhimurium/sh-INHA may provide a novel cancer treatment option.

Analysis of genes responding to ultraviolet B irradiation of HaCaT keratinocytes using a cDNA microarray.

BACKGROUND: Ultraviolet (UV) B irradiation causes many important biological changes in skin, which lead to pathophysiological alterations of the homeostatic environment. OBJECTIVES: To gain more insight into the molecular events provoked by UVB irradiation, we performed cDNA microarray analysis. METHODS: Immortalized HaCaT keratinocytes were irradiated with a high cytotoxic dose of UVB (50 mJ cm(-2)), and total RNA was isolated. Fluorescently labelled probes were prepared by reverse transcription and were hybridized with cDNA microarray slides made using 840 cDNA clones. RESULTS: Time-course cDNA microarray analysis revealed the global gene expression profile after UVB exposure. Of 840 genes tested, 192 genes showed changes in their expression levels at one or more of four time points. The genes were clustered into four groups according to their expression patterns in a self-organizing maps analysis. Classification of these genes into nine functional categories revealed that UVB irradiation affected several biological processes. The genes that were first upregulated and then returned to normal levels included several genes related to the inhibition of cell growth and the proteasome pathway. Conversely, the expressions of many genes involved in the cytoskeleton, signal transduction, metabolism and transcription were first downregulated or unchanged and then upregulated later, reflecting the recovery of UVB-damaged cellular activities. CONCLUSIONS: These results demonstrate the complexity of the transcriptional profile of the UVB response, and provide a basis for the global characterization of UV-regulated gene expression.

Microtubule disruption in keratinocytes induces cell-cell adhesion through activation of endogenous E-cadherin.

The association of the cytoskeleton with the cadherin--catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin-catenin-actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.

Role of reconstituted basement membrane in human granulosa cell culture.

The effects of Matrigel, a reconstituted basement membrane, on human granulosa cells were investigated. Cells were obtained from follicular aspirate in the course of oocyte retrieval for in vitro fertilization and were cultured on either a surface coated with Matrigel or uncoated plastic. Light and electron microscopy showed that granulosa cells cultured on Matrigel demonstrated three-dimensional aggregated cells with well differentiated morphology: numerous lipid droplets, microvilli, junctional complexes and lumen-like structures were seen. In contrast, cells cultured on plastic were flattened, poorly differentiated and showed apoptotic cells. Immunocytochemistry showed that the proportion of immunopositive cells for 3beta-hydroxysteroid dehydrogenase was increased in cultures on Matrigel. The results of the present study suggest that culture on Matrigel promotes the differentiation of human granulosa cells and provides a useful tool which may improve the efficiency of in vitro fertilization.

Disassembly of focal adhesions during apoptosis of endothelial cell line ECV304 infected with Orientia tsutsugamushi.

Many bacterial pathogens induce apoptosis in their host cells. We observed the cellular effect of ECV304 cells infected with Orientia tsutsugamushi. The infected cells became rounded and floated in culture supernatant. These floating cells as well as adherent cells exhibited typical features of apoptosis, such as DNA fragmentation and TUNEL staining. As many cells detached from growth substrate, we examined the focal adhesion using the immunofluorescence assay method and observed decreased focal adhesions in heavily infected cells. As endothelial cells could undergo apoptosis by the loss of focal adhesions, this change of focal adhesions may account for the Orientia-induced apoptosis.

Apoptosis of endothelial cell line ECV304 persistently infected with Orientia tsutsugamushi.

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase-mediated dUTP nick end-labeling reaction and DNA fragmentation. We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL-6 and IL-8. These results show the first evidence of in vitro-persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.

Molecular and serologic survey of Orientia tsutsugamushi infection among field rodents in southern Cholla Province, Korea.

Field rodents were collected from six areas in southern Cholla Province, Korea from October to December 1993. Twenty-eight (24%) of the 119 Apodemus agrarius were seropositive (> 1:10) for Orientia tsutsugamushi by the passive hemagglutination assay (PHA). Of the seropositive cases, 11 specimens had antibody titers greater than 1:80. No seropositive specimens were found among the eight Crocidura lasiura collected. On the other hand, the polymerase chain reaction (PCR) amplified about 520 basepairs of a gene encoding the 56-kD protein from the genomic DNA of 12 strains of O. tsutsugamushi tested. This target DNA sequence was amplified from the 11 (8.7%) blood specimens of A. agrarius, and one of the eight C. lasiura also showed evidence of O. tsutsugamushi infection by PCR. Only one of the PCR-positive samples was also PHA-positive. These results suggest that the PCR combined with a serologic assay more accurately detects the degree of infection of rodents with rickettsiae-causing scrub typhus in epidemiologic surveys.

Identification of antigenic differences between the phosphorylated and nonphosphorylated forms of the E7 protein of human papillomavirus type 16.

To analyze the antigenic properties of the human papillomavirus type 16 E7 oncoprotein, two monoclonal antibodies, VD6 and IB10, that have different reactivities to the E7 protein were generated. While the VD6 antibody reacted strongly with E7 protein in CaSki cell extracts, the other antibody, IB10, showed much weaker reactivity with E7. This reactivity increased in a dose-dependent manner in the presence of the casein kinase II-specific inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). Antigenic site estimation and an in vitro phosphorylation assay, using bacterially expressed E7 protein, demonstrated that the weak reactivity of IB10 was related to the phosphorylation status of the E7 protein. Phosphorylation of E7 reduced considerably the reactivity of IB10 but did not affect the reactivity of VD6, which reacts with the N-terminal portion of E7. In immunoprecipitation (IP) assays, IB10 precipitated weakly the E7 protein from CaSki cell extracts. Together, these data suggest that unphosphorylated E7 protein shows distinct antigenic character compared to its phosphorylated form under denaturing conditions; however, under native conditions, the phosphorylated and nonphosphorylated E7 proteins have some antigenic cross-reactivity.

Infection rate of Leptospira interrogans in the field rodent, Apodemus agrarius, in Korea.

Leptospirosis has significantly decreased in Korea since 1988, following the leptospiral vaccination programme initiated in 1988. Whether this wholly explains the decreased incidence is uncertain. As an initial step to answer this question, infection rates of Leptospira interrogans in field rodents, Apodemis agrarius, were examined and compared with previous data. Two hundred and twenty-two A. agrarius were captured during October-December 1996. Spirochaetes were isolated from 22 (9.9%) and leptospiral DNA was detected in an additional 6 rodents (12.6%). Subsequent microscopic agglutination tests (MAT) classified all these isolates as L. interrogans serogroup Icterohaemorrhagiae serovar lai. The above data did not significantly differ from previous surveys in 1984-7. There was no significant change of L. interrogans infection in field rodents following the introduction of the vaccination programme in Korea. Further studies are needed to determine the role of human vaccination in reducing incidence.

Long-term oral administration of ginseng extract decreases serum gamma-globulin and IgG1 isotype in mice.

Effects of long-term oral administration of ginseng extract on serum protein profile and immunoglobulin (Ig) isotypes were studied in mice. Ginseng extract was orally administered to healthy female mice for 52 days at doses of 30 and 150 mg/kg per day and serum protein electrophoretograms and Ig isotypes levels were evaluated. Serum level of gamma-globulin was decreased dose dependently to 82% (P < 0.05) and 56% (P < 0.01) of control values at the doses of 30 and 150 mg/kg per day, respectively. Levels of total protein, albumin, alpha2- and beta-globulin fractions, as well as the ratio of albumin to globulin (A/G) did not change significantly. However, the alpha1-globulin level increased by 24% (P < 0.05) at the doses of 30 and 150 mg/kg per day. Among the Ig isotypes, including IgG1, IgG2a, IgG2b, IgG3, IgM and IgA, serum IgG1 was dose dependently decreased to 68% (P < 0.05) of control values at the dose of 150 mg/kg per day without significant changes in other Ig isotypes. As IgG1 isotype is rarely cytotoxic and can act as a blocking antibody, it is suggested that the selective decrease in serum IgG1 induced by ginseng extract without changes in the cytotoxic antibodies such as IgG2a may be helpful for the prevention and inhibition of cancer.

Major sequence variants in E7 gene of human papillomavirus type 16 from cervical cancerous and noncancerous lesions of Korean women.

Geographic specificity of nucleotide sequence variations in the coding and noncoding regions of HPV 16 genome has been reported. Little has been known, however, regarding whether these naturally occurring sequence variations of HPV 16 may result in marked differences in biological properties, such as oncogenic potential. This study was performed to identify sequence variants in the HPV 16 E7 gene derived from Korean women with cervical cancerous and noncancerous lesions, and to assess the association between the sequence variant and the cervical cancer. We examined E7 variants of HPV 16 in a total of 157 patients with no cervical disease (NCD, n = 87) or cervical neoplasia (cervical intraepithelial neoplasia 3, n = 21; cervical carcinoma, n = 49), using the nested polymerase chain reaction (PCR) and the PCR-directed sequencing methods with outer consensus and inner type-specific primers. Forty-two (NCD, n = 9; CIN 3, n = 6; cervical carcinoma, n = 27) of 157 cervical samples contained HPV 16 E7 DNA, but only 8 had prototype sequences. Four variants of the HPV 16 E7 gene were identified. The variant with a single nucleotide change at position 647 (A --> G, Asn --> Ser) was found in about 60% of DNA samples with HPV 16. The second most common variant, found in 16.7% of cases, had three silent mutations at positions 732 (T --> C), 789 (T --> C), and 795 (T --> G). Two other variants were detected, one in a patient with cervical cancer and the other in a patient with no cervical disease. One had a single nucleotide change at position 666 (G --> A) and the other had one silent mutation at position 796 (T --> C). The most common variant in Korea has a change of nucleotide affecting the predicted amino acid related with high antigenicity and binding to retinoblastoma protein. There was a statistically significant trend for this variant to be more frequently detected in cancerous lesions of the uterine cervix than in noncancerous lesions. These data suggest that naturally occurring sequence variants of HPV 16 E7 gene may have different oncogenic properties.

Genetic analysis of Borrelia burgdorferi sensu lato in Korea using genomic hybridization and 16S rRNA gene sequence determination.

Nine Borrelia burgdorferi sensu lato isolated in Korea were subjected to genomic hybridization using 16S rRNA gene probe and specific restriction patterns (HindIII and EcoRV) led these nine Borrelia into five subtypes. The evolutionary relationships of the five isolates corresponding to five RFLP groups were measured through the sequence determination of 16S rRNA gene and phylogenetic analysis. The isolates 935T (group I), 934U and 17Y (Group IIa, IIb) were well clustered with B. garinii and B. afzelii. 5MT and 9MT strains (Group IIIa and Group IIIb) formed a common branch shared with B. afzelii cluster although the evolutionary distance was rather long. So, most of B. burgdorferi sensu lato in Korea was B. afzelii or B. afzelii-related group and some minor group such as B. garinii also existed.

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.

Detection of leptospiral DNA by PCR.

An EcoRI fragment (1.2 kb) which is highly conserved among Leptospira interrogans isolated in Korea was cloned into pBluescript vector from L. interrogans serovar lai WH20. The EcoRI fragment was sequenced, and a pair of primers (LP1 and LP2) was designed for PCR assay. PCR amplification of target DNA obtained from cultured L. interrogans showed that 274 bp could be detected when as little as 100 fg of leptospiral genomic DNA was used in the reaction mixture. No amplification of DNA was detected from DNA of Leptospira biflexa serovars patoc and sau paulo, Borrelia burgdorferi, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium. Amplification of 274-bp target DNA could be detected in DNA samples purified from 500 microliters of blood collected from experimentally infected gerbils 2 days after infection, while antibodies to L. interrogans could be detected by the microscopic agglutination test 7 days after infection. The specificity and high sensitivity of the test provided valuable tools for the early diagnosis of leptospirosis.