Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Creation of immortalised epithelial cells from ovarian endometrioma.

BACKGROUND: Epithelial cells of endometriotic tissues are difficult to propagate in vitro as experimental material is scarce owing to their limited life span. However, there is an increasing concern regarding their malignant transformation in ovaries. The present study sought to generate their stable culture system. METHODS AND RESULTS: Purified epithelial cells isolated from ovarian endometriomas using microscopic manipulation were successfully immortalised by combinatorial transfection of human cyclinD1, cdk4 and human telomerase reverse transcriptase (hTERT) genes, whereas the introduction of hTERT alone, or together with cdk4, was insufficient for immortalisation, leading to cellular senescence. We confirmed stable cytokeratin expression in the immortalised cells, proving their epithelial origin. These cells expressed progesterone receptor B and showed significant growth inhibition by various progestins. Oestrogen receptor (ER) expression was detected in these cells, albeit at low levels. Additional overexpression of ERα generated stable cells with oestrogen-dependent growth activation. Soft-agar colony formation assay and nude mice xenograft experiments demonstrated that these cells, even those with additional inactivation of p53, did not have transformed phenotypes. CONCLUSION: We for the first time generated immortalised epithelial cells from ovarian endometrioma that retained sex steroid responsiveness. These cells are invaluable tools not only for the consistent in vitro work but also for the study of molecular pathogenesis or carcinogenesis of endometriosis.

Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification.

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.

CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential.

In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to < 30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.

Carrier cell-based delivery of replication-competent HSV-1 mutants enhances antitumor effect for ovarian cancer.

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. We have developed replication-competent attenuated herpes simplex virus type 1 (HSV-1) mutants, named HF10 and Hh101, which have been evaluated for their oncolytic activities. However, the host immune system remains a significant obstacle to effective intraperitoneal administration of these viruses in the clinical setting. In this study, we investigated the use of these HSV-1 mutants as oncolytic agents against ovarian cancer and the use of human peritoneal mesothelial cells (MCs) as carrier cells for intraperitoneal therapy. MCs were efficiently infected with HSV-1 mutants, and MCs loaded with HSV-1 mutants caused cell killing adequately when cocultured with cancer cells in the presence or absence of HSV antibodies. In a mouse xenograft model of ovarian cancer, the injection of infected carrier cells led to a significant reduction of tumor volume and prolonged survival in comparison with the injection of virus alone. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on ovarian cancer, which may be further enhanced by the utilization of a carrier cell delivery system, based on amplification of viral load and possibly on avoidance of neutralizing antibodies.

Expression of HER-2 affects patient survival and paclitaxel sensitivity in endometrial cancer.

BACKGROUND: Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K-AKT and MAPK-ERK signalling, as well as drug sensitivity. METHODS AND RESULTS: Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor. Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways. Short interfering RNA (siRNA)-based knockdown of HER-2 in endometrial cancer cells led to a significant reduction in phosphorylated AKT (p-AKT) expression, indicating the existence of a HER-2/PI3K-AKT axis. As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel. Short interfering RNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form. CONCLUSION: HER-2 is a significant prognostic factor of endometrioid-type endometrial cancer, as well as a key molecule that affects paclitaxel sensitivity by HER-2 interaction with the PI3K-AKT pathway.

Effect of intrasplenic transplantation of immortalized human hepatocytes in the treatment of acetaminophen-induced acute liver failure SCID mice.

OBJECTIVE: We have established immortalized human hepatocytes by transduction of HPV16 E6/E7 and hTERT (HHE6E7T-1). The cells retained the characteristics of differentiated hepatocytes, but the functional characteristics such as albumin secretion, ureogenesis, and glyconeogenesis decreased gradually as the passages progressed beyond 200 population doublings (PDs). In this report, we transplanted minimally differentiated HHE6E7T-1 cells into the spleens of acute liver failure severe combined immunodeficiency (SCID) mice to examine the potential of the cells to redifferentiate in vivo. MATERIALS AND METHODS: Acute liver failure was induced in SCID mice by intraperitoneal injection of 400 mg/kg acetaminophen. Two hours later, HHE6E7T-1 cells at 200 PDs were transplanted: group 1 (n = 10) 50 microL phosphate-buffered saline (PBS); group 2 (n = 9), lysate of 1 x 10(6) HHE6E7T-1 cells at 200 PDs resuspended in 50 microL PBS; and group 3 (n = 8), 1 x 10(6) HHE6E7T-1 cells. Survival rates at 7 days after transplantation were compared. Blood glucose levels, plasma ammonia levels, and spleen histology were examined at 24 hours after transplantation. RESULTS: Survivals in each group were: 30% for group 1, 33% for group 2, and 100% for group 3. The survival of group 3 was significantly higher than groups 1 or 2 (P < .01). Plasma ammonia levels in group 3 (200 +/- 34 microg/dL) were significantly lower than those in group 1 (325 +/- 92 microg/dL; P < .05). Blood glucose levels in group 3 (110 +/- 20 mg/dL) were significantly higher than those in group 1 (83 +/- 14 mg/dL; P < .05). Upon histologic examination of spleen, the clusters of HHE6E7T-1 cells were clearly identified. CONCLUSIONS: The immortalized human hepatocytes, HHE6E7T-1 at 200 PDs, improved the survival of acute liver failure mice through possible redifferentiation in vivo.

Wild-type p53 and p73 negatively regulate expression of proliferation related genes.

When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or beta-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21.

Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo.

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF( E6-EGFP ) cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF( E6-EGFP ) cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF( E6-EGFP ) cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF( E6-EGFP ) cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF( E6-EGFP ) cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes.

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements.

Several genetic mutations have been identified in human endometrial cancers, but the specific combinations of mutations required to form endometrial cancer cells remain unknown. In the present study, we established an in vitro model of endometrial carcinogenesis, in which defined genetic elements were introduced into endometrial epithelial cells to create transformed endometrial cells at different stages. Introduction of the human papillomavirus type 16 E6/E7 gene and the human telomerase reverse transcriptase (hTERT) gene into human primary endometrial epithelial cells was sufficient to generate immortalized cells. Introduction of hTERT in early passages stabilized telomeres and created immortalized cells with normal karyotype, whereas introduction of hTERT in later passages generated immortalized cells but with widespread chromosome abnormalities. However, neither of those two immortalized cell lines exhibited tumorigenic phenotypes. Tumorigenic endometrial epithelial cells with invasive capacity were created by introducing a mutant K-ras allele into immortalized cells, keeping their chromosomes intact. Inhibiting the PTEN gene and activating Akt pathways did not create tumorigenic phenotypes, although the latter conferred anchorage-independent growth capacity. These findings suggest that neoplastic transformation of human endometrial cells can occur in the absence of widespread chromosomal abnormality, and that the combination of Rb inactivation, telomerase activation and altered K-ras signaling is sufficient for in vitro neoplastic transformation. The present experimental model can help clarify the genetic requirements for endometrial carcinogenesis, and it is useful for testing and developing specific inhibitors of specific oncogenic pathways.

Establishment of an immortalised human ovarian surface epithelial cell line without chromosomal instability.

Epithelial ovarian carcinoma is thought to derive from ovarian surface epithelium (OSE). The black box of the early molecular changes in ovarian carcinogenesis is being interpreted by the development of experimental systems employing immortalised human OSE cells. However, the existing cell lines of the OSE cells have limited utility due to chromosomal instability. Our goal was to establish new immortalised human OSE cells that retain the original characteristics of the primary cells without chromosomal alterations. Using primary human OSE cells obtained from a postmenopausal patient with endometrial cancer, five cell lines ('HOSE1' lines) were newly established by infection with retroviral expression vectors containing type 16 human papillomavirus (HPV-16) E6, E7, a variant E6 (E6delta151), and Bmi1 polycomb gene, in combination with telomerase reverse transcriptase (hTERT). Consequently, five HOSE1s cell lines, HOSE1s-E6/hTERT, -E7/hTERT, -E6/E7/hTERT, -E6delta151/E7/hTERT, and -E6delta151/Bmi1/hTERT, grew beyond the population doubling number of 200. These cell lines, except for HOSE1-E6/hTERT, essentially showed the original features of the primary human OSE cells. Of them, HOSE1-E7/hTERT preserved diploidy in a kariotype analysis, and did not show transformed phenotypes in anchorage-independent growth and tumour formation. Thus, HOSE1-E7/hTERT may provide a novel model system with which to investigate the mechanisms of early molecular changes.

The human papillomavirus E6 and E7 inducible oncogene, hWAPL, exhibits potential as a therapeutic target.

Here we show that human papillomavirus (HPV) E6 and E7 oncoproteins induce hWAPL expression. In addition, small interfering RNA (siRNA) of hWAPL suppressed the growth of tumours derived from SiHa cells in nude mice. Thus, hWAPL may be one of the effective targets of uterine cervical cancer therapy.

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways.

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis.

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.

Cementum matrix formation in vivo by cultured dental follicle cells.

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation.

Karyotype and fluorescence in situ hybridization analyses have demonstrated the frequent presence of an altered static state of the number of chromosomes (ie, aneuploidy) in lung cancer, but it has not been directly established whether aneuploidy is in fact associated with a persistent increase in the rate of chromosomal losses and gains (ie, chromosome instability, or CIN). The study presented here used a panel of 10 lung cancer cell lines to provide for the first time direct evidence that CIN is a common feature in lung cancer cell lines in association with the presence of significant aneuploidy. In addition, we found that the CIN phenotype correlates well with the presence of p53 mutations. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactivation of p53 itself is unlikely to directly induce CIN in lung cancer cells. Interestingly, however, significant CIN could be induced in conjunction with the generation of aneuploid populations when the mitotic spindle formation was transiently abrogated in p53-inactivated cells. These results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cycle under certain forms of mitotic stresses, which would result in the induction of the CIN phenotype in conjunction with the generation of aneuploidy.

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells.

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2-*) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2',7'-dichlorofluorescin (DCFH) into 2',7'-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2-*, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (* OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2-*-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53(Val138) induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2-*. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2-*, but not its downstream compounds H2O2 or * OH.

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells.

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.