Using lactic acid bacteria and packaging with grapefruit seed extract for controlling Listeria monocytogenes growth in fresh soft cheese.

Various cheese products are involved in outbreaks of listeriosis worldwide due to high consumption and prolonged refrigerated storage. The objective of this study was to determine the efficacy of using lactic acid bacteria and packaging with grapefruit seed extract (GSE) for controlling Listeria monocytogenes growth in soft cheese. Leuconostoc mesenteroides and Lactobacillus curvatus isolated from kimchi were used as a starter culture to make a soft cheese, which was inoculated with a cocktail strain of L. monocytogenes. The soft cheese was packed with low-density polyethylene, biodegradable polybutylene adipate-co-terephthalate (PBAT), low-density polyethylene with GSE, or PBAT with GSE and stored at 10°C and 15°C. Leuconostoc mesenteroides (LcM) better inhibited the growth of L. monocytogenes than Lb. curvatus. The PBAT with GSE film showed the best control for the growth of L. monocytogenes. When both LcM and PBAT with GSE were applied to the soft cheese, the growth of L. monocytogenes was inhibited significantly more than the use of LcM or PBAT with GSE alone. In all test groups, water activity, pH, and moisture on a fat-free basis decreased, and titratable acidity increased compared with the control group. These results suggest that LcM isolated from kimchi and PBAT with GSE packaging film can be used as a hurdle technology to lower the risk of L. monocytogenes in soft cheese at the retail market.

Single-stage cell-based cartilage repair in a rabbit model: cell tracking and in vivo chondrogenesis of human umbilical cord blood-derived mesenchymal stem cells and hyaluronic acid hydrogel composite.

OBJECTIVE: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have gained popularity as a promising cell source for regenerative medicine, but limited in vivo studies have reported cartilage repair. In addition, the roles of MSCs in cartilage repair are not well-understood. The purpose of this study was to investigate the feasibility of transplanting hUCB-MSCs and hyaluronic acid (HA) hydrogel composite to repair articular cartilage defects in a rabbit model and determine whether the transplanted cells persisted or disappeared from the defect site. DESIGN: Osteochondral defects were created in the trochlear grooves of the knees. The hUCB-MSCs and HA composite was transplanted into the defect of experimental knees. Control knees were transplanted by HA or left untreated. Animals were sacrificed at 8 and 16 weeks post-transplantation and additionally at 2 and 4 weeks to evaluate the fate of transplanted cells. The repair tissues were evaluated by gross, histological and immunohistochemical analysis. RESULTS: Transplanting hUCB-MSCs and HA composite resulted in overall superior cartilage repair tissue with better quality than HA alone or no treatment. Cellular architecture and collagen arrangement at 16 weeks were similar to those of surrounding normal articular cartilage tissue. Histological scores also revealed that cartilage repair in experimental knees was better than that in control knees. Immunohistochemical analysis with anti-human nuclear antibody confirmed that the transplanted MSCs disappeared gradually over time. CONCLUSION: Transplanting hUCB-MSCs and HA composite promote cartilage repair and interactions between hUCB-MSCs and host cells initiated by paracrine action may play an important role in cartilage repair.

Do Contrast-Fill Patterns Immediately After Coil Embolization of Small Saccular Aneurysms Impact Long-Term Results?

PURPOSE: It is generally accepted that filling of a saccular aneurysm with contrast immediately after coil embolization predisposes to later recanalization. However, not all such scenarios evolve similarly over time. We investigated outcomes of small (≤ 7 mm) aneurysms with contrast-filled sacs immediately after coil embolization, evaluating the impact of pattern and degree of filling on subsequent recanalization. METHODS: Between January, 2008 and December, 2010, 186 small (≤ 7 mm) saccular aneurysms that retained contrast after coil embolization accrued for this study. Lesions were categorized by pattern (eccentric vs. concentric) and degree of filling on working projections. Clinical and morphologic factors were also analyzed to assess impact on subsequent recanalization. Morphologic outcomes at 6 months or more were assessed. RESULTS: In 93.5 % (174/186) of aneurysms with visible contrast retention, complete occlusion was evident on follow-up imaging studies at 6 months. Multiple logistic regression analysis indicated that eccentric (vs. concentric) contrast filling carried greater risk of subsequent recanalization (p = 0.020). Stent placement and progressive occlusion were also linked, falling short of statistical significance (p = 0.089). Of 166 progressively occluded aneurysms followed for more than 12 months (mean, 30.8 ± 7.3 months), 158 (95.2 %) exhibited stable occlusion. CONCLUSION: Small (≤ 7 mm) aneurysms that retain contrast immediately after coil embolization are more likely to become completely occluded over time through progressive thrombosis. However, an eccentric fill pattern may predispose to recanalization.

Fate of Coiled Aneurysms with Minor Recanalization at 6 Months: Rate of Progression to Further Recanalization and Related Risk Factors.

BACKGROUND AND PURPOSE: Minor recanalization in coiled aneurysms may remain stable with time or may progress to major recanalization. Our aim was to monitor the aneurysms displaying minor recanalization in imaging studies at 6 months, gauging major recanalization rates and related risk factors through extended follow-up. MATERIALS AND METHODS: Sixty-five aneurysms (in 65 patients) showing minor recanalization in follow-up imaging at 6 months were reviewed retrospectively. Medical records and radiologic data accruing during extended monitoring (mean, 24.8 ± 8.2 months) were assessed. Univariate and multivariate analyses were conducted to identify risk factors for progression from minor-to-major recanalization. RESULTS: Progression to major recanalization was observed in 24 (36.9%) of the initially qualifying aneurysms during a follow-up of 112.5 aneurysm-years, for an annual rate of 17.84% per aneurysm-year. Progression was determined chronologically as follows: 14 (58.3%) at 6 months, 8 (33.3%) at 18 months, and 2 (8.4%) at 30 months. Stent deployment significantly decreased the occurrence of major recanalization (OR = 0.22, P = .03), whereas antiplatelet therapy (OR = 0.82, P = .75), posterior location (OR = 0.24, P = .20), and second coiling for recanalized aneurysms (OR = 0.96, P = .96) were unrelated. CONCLUSIONS: Our analysis determined a 36.9% rate of major recanalization during a follow-up of 112.5 aneurysm-years in coiled aneurysms showing minor recanalization at 6 months. Stent deployment alone conferred a protective effect, preventing further recanalization without additional treatment. Given the fair probability of late major recanalization, aneurysms showing minor recanalization at 6 months should be monitored diligently, particularly in the absence of stent placement.

Properties of poly(lactide)-coated paperboard for the use of 1-way paper cup.

Poly(lactide)-coated paperboards were prepared by a solution coating method, and the effect of coating to improve properties of paperboard used for the manufacturing of 1-way paper cups was tested. Surface of PLA-coated paperboards was smooth and shiny like PE-coated paperboard, and the coating weight and thickness increased linearly with increasing PLA concentration of coating solution. Tensile strength (TS) and elongation at break (E) of the paperboard also increased after PLA coating. Water vapor barrier or water-resistant properties tested, such as water vapor permeability (WVP), water absorptiveness (WA), and contact angle (CA) of water drop, indicated that water resistance of the paperboard was improved through surface coating with PLA. The increase in water resistance of PLA-coated paperboards was mainly due to the hydrophobicity of PLA and the improvement of water barrier properties increased depending on the PLA concentration. In addition, PLA-coated paperboard showed strong heat sealing property when coated with more than 1 w/v% of PLA. Wet strength of PLA-coated (3, w/v%) paperboard was comparable to or greater than that of PE-coated paperboard. All the test results indicated that the PLA-coated paperboard can be exploited for the manufacturing of 1-way paper cups as an alternative to the PE-coated paperboard.

Prostate cell cultures as in vitro models for the study of normal stem cells and cancer stem cells.

Current existing therapies for prostate cancer eradicate the majority of cells within a tumor. However, most patients with advanced cancer still progress to androgen-independent metastatic disease that remains essentially incurable by current treatment strategies. Recent evidence has shown that cancer stem cells (CSCs) are a subset of the tumor cells that are responsible for initiating and maintaining the disease. Understanding normal stem cells and CSCs may provide insight into the origin of and new therapeutics for prostate cancer. Normal stem cells and CSCs have been identified in prostate tissue by the use of several markers or techniques. Although research on stem cells has been limited by the lack of suitable in vitro systems, recent studies show that not only primary cells but also several established cell lines may exhibit stem cell properties. This review discusses various in vitro culture systems to propagate normal prostate stem cells and prostate CSCs together with molecular markers. These in vitro cell culture models should be useful for elucidating the differentiation of prostatic epithelium and the biological features of prostate cancer.

Characterization of the androgen receptor in a benign prostate tissue-derived human prostate epithelial cell line: RC-165N/human telomerase reverse transcriptase.

The majority of prostate epithelial cell lines stably expressing wild-type (wt) or mutant (mt) androgen receptor (AR) are derived from metastatic prostate cancers. Therefore, the wt AR-expressing RC-165N/human telomerase reverse transcriptase (hTERT) cell line derived from the benign prostate tissue of an African-American patient provides a unique opportunity to assess the functional status of AR in a cellular context not studied before. Although androgen-induced expression of known androgen responsive genes such as PMEPA1, and NDRG1 was observed in RC-165N/hTERT, this cell line expresses prostate-specific antigen (PSA) at significantly lower levels. Chromatin immunoprecipitation assay revealed androgen-dependent binding of AR to androgen response elements of PSA, PMEPA1 and NDRG1 genes. Similarities, as well as differences were noted in the expression of androgen responsive genes between RC-165N/hTERT and LNCaP cells. Comprehensive evaluations of AR functions in RC-165N/hTERT cells suggest that whereas some features of known AR functions are maintained in this benign prostatic tissue-derived cell line, other AR functions are not retained. Objective evaluations of similar cell lines will lead to the understanding of AR functions in prostate growth and differentiation.

Fryns syndrome phenotype caused by chromosome microdeletions at 15q26.2 and 8p23.1.

BACKGROUND: Fryns syndrome (FS) is the commonest autosomal recessive syndrome in which congenital diaphragmatic hernia (CDH) is a cardinal feature. It has been estimated that 10% of patients with CDH have FS. The autosomal recessive inheritance in FS contrasts with the sporadic inheritance for the majority of patients with CDH and renders the correct diagnosis critical for accurate genetic counselling. The cause of FS is unknown. METHODS: We have used array comparative genomic hybridisation (array CGH) to screen patients who have CDH and additional phenotypic anomalies consistent with FS for cryptic chromosome aberrations. RESULTS: We present three probands who were previously diagnosed with FS who had submicroscopic chromosome deletions detected by array CGH after normal karyotyping with G-banded chromosome analysis. Two female infants were found to have microdeletions involving chromosome band 15q26.2 and one male had a deletion of chromosome band 8p23.1. CONCLUSIONS: We conclude that phenotypes similar to FS can be caused by submicroscopic chromosome deletions and that high resolution karyotyping, including array CGH if possible, should be performed prior to the diagnosis of FS to provide an accurate recurrence risk in patients with CDH and physical anomalies consistent with FS.

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas.

SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

Detection of cancer cells in peripheral blood of stomach cancer patients using RT-PCR amplification of tumour-specific mRNAs.

BACKGROUND: RT-PCR amplification of tumour-specific mRNA has been used for the detection of cancer cells in peripheral blood. AIM: To evaluate the characteristics of the tumour specific mRNA species in peripheral blood of stomach cancer patients. METHODS: We analysed CEA, GalNAc-T, MUC-1, c-MET and hTERT mRNA expression in the stomach cancer cell lines and tissues, lymph nodes and peripheral blood of stomach cancer patients using RT-PCR. RESULTS: In RT-PCR analysis of the peripheral blood, 4%, 8%, 21%, 46%, and 100% of stomach cancer patients were positive for CEA, GalNAc-T, c-MET, hTERT and MUC-1 mRNA, respectively, but MUC-1 mRNA was also positive in all normal blood samples. The detection of hTERT mRNA was correlated with poor differentiation (P = 0.01) and lymph node metastasis (P = 0.009). The presence of c-MET mRNA was correlated with T stage (P = 0.025), lymph node metastasis (P = 0.036), distant metastasis (P = 0.031), and stage of the stomach cancer (P = 0.023). CONCLUSIONS: Our study suggest that hTERT mRNA in peripheral blood can be a molecular marker for gastric cancer. We also showed that each molecular marker can be correlated with the clinicopathological features of the patients.

A novel human cancer culture model for the study of prostate cancer.

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. We have successfully established an immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor with telomerase. The actively proliferating early passaged RC-58T cells were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase was detected in RC-58T/hTERT cells but not RC-58T cells. RC-58T/hTERT cells are currently growing well at passage 50, whereas RC-58T cells senesced at passage 7. RC-58T/hTERT cells exhibit transformed morphology. More importantly, these immortalized cells showed anchorage-independent growth as they formed colonies in soft agar and grew above the agar layer. Expression of androgen-regulated prostate specific gene NKX3.1 and epithelial specific cytokeratin 8 (CK8) but not prostate specific antigen (PSA) and androgen receptor was detected in RC-58T/hTERT cells. Prostate stem cell antigen (PSCA) and p16 were also expressed in this cell line. RC-58T/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1 known potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes Y, 3p, 10p, 17p, 18q and the gain of chromosomes 16 and 20. These results demonstrate that this primary tumor-derived HPE cell line retained its transformed phenotypes and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of an established human prostate cancer cell line from a primary tumor of a prostate cancer patient with telomerase.

Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood.

PURPOSE: The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions. MATERIAL AND METHODS: In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis. RESULT: The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cells in 1 ml of peripheral blood. CONCLUSION: The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.

A novel human cell culture model for the study of familial prostate cancer.

Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells. 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5. 957E/hTERT cells exhibit epithelial morphology. Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells. Prostatic stem cell antigen and p16 were also expressed in this line. 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth. Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range. However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q. The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20. The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes. This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient.

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway.

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.

Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors.

Critical events in prostate tumorigenesis and metastasis likely include the abnormal activation and expression of specific genes. Using RNA expression profiling techniques, we have identified a transcript originating from the activated in prostate cancer (AIPC) gene, the expression of which is preferentially up-regulated in several cultured prostate tumor cell lines and human primary prostate tumors. Sequence analysis revealed that the AIPC protein encodes six PDZ domains, which are protein-protein binding domains likely involved in protein clustering and scaffolding. Immunohistochemical analysis of a tissue microarray comprising 158 tumor, 18 high-grade prostatic intraepithelial neoplasia, and 91 normal prostate specimens with an anti-AIPC antibody demonstrated abundant AIPC protein expression in 75% of tumors, 83% of prostatic intraepithelial neoplasia lesions, and 3% of normal tissues (P < 0.0001). These data suggest that the accumulation of AIPC protein may be closely associated with the initiation or early promotion of prostate tumorigenesis.

Immortalization of rat middle ear epithelial cells by adeno 12-SV40 hybrid virus.

Rat middle ear epithelial cells were infected with the adeno 12-SV40 hybrid virus. The cell line thus obtained displays features of primary cultured epithelial cells in both light microscopic and ultrastructural examinations. The immortalized cells have been in continuous proliferation for 40 passages and more than 17 months. Immunohistochemical analysis of the immortalized cells was positive for the SV40 T antigen and the tumor suppressor protein p53. The cells also stained positive for cytokeratin, an epithelial cell marker, and negative for vimentin, a fibroblast marker. These results, together with karyotype analysis, indicate that this cell line originated from rat middle ear epithelial cells and retains the characteristics of epithelial cells. This cell line will be useful for studying the normal cellular biology of middle ear epithelial cells, as well as the cellular and molecular mechanisms involved in the bacteria-middle ear epithelial cell interaction.

Molecular and genetic mechanisms of prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy in American men and is the second leading cause of cancer death in males in the United States. Despite its high incidence, the molecular and genetic events involved in progression of prostate cancer remain poorly understood. In vitro models of human prostate epithelial (HPE) cells provide a practical approach to the analysis of the molecular and genetic mechanisms underlying prostate carcinogenesis. We reported the immortalization of normal adult HPE cells by transfection of the HPV-18 DNA and the subsequent conversion of such nontumorigenic but immortalized cells (HPV-18 C-1) into tumorigenic cells by the introduction of an activated Kras oncogene. Recently, we have demonstrated the malignant transformation of HPV-18 C-1 cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea (NMU). Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No TP53 or RAS mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10(p10>pter) and gain of the q arm of chromosome 8 (p10>ptr) (the most frequent cytogenetic changes observed directly in prostate cancer patients) were observed only in the tumor outgrowths. These findings provide the first evidence of malignant transformation of HPE cells exposed to a chemical carcinogen.

RNA damage and inhibition of neoplastic endothelial cell growth: effects of human and amphibian ribonucleases.

Angiogenesis defines the many steps involved in the growth and migration of endothelial cell-derived blood vessels. This process is necessary for the growth and metastasis of tumors, and considerable effort is being expended to find inhibitors of tumor angiogenesis. This usually involves screening of potential anti-angiogenic compounds on endothelial cells. To this end, two candidate anti-angiogenic RNA-damaging agents, onconase and (-4)rhEDN, were screened for their effects on endothelial cell proliferation using three distinct types of endothelial cells in culture: HPV-16 E6/E7-immortalized human umbilical vein endothelial cells (HUVECs), a Kras-transformed HPV-16 E6/E7 HUVEC (Rhim et al., Carcinogenesis 4, 673-681, 1998), and primary HUVECs. Onconase similarly inhibited proliferation in all three cell lines (IC(50) = 0.3-1.0 microM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC(50) = 0.02-0.06 microM) than on primary HUVECs (IC(50) > 0.1 microM). Differential sensitivity to these agents implies that more than one endothelial cell type must be used in proliferation assays to screen for novel anti-angiogenic compounds.

Vitamin D resistance in RAS-transformed keratinocytes: mechanism and reversal strategies.

Human retinoid X receptor alpha (hRXRalpha) plays a critical role in DNA binding and transcriptional activity through its heterodimeric association with several members of the nuclear receptor superfamily, including the vitamin D receptor (VDR). Several cancer cell lines derived from different tissues have been shown to be resistant to the growth-inhibitory action of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the biologically active metabolite of vitamin D(3). Here we show that in RAS-transformed keratinocytes, Ser260 of hRXRalpha is phosphorylated through the RAS-RAF-MAP kinase cascade. This phosphorylation event results in the inhibition of vitamin D signaling via VDR/hRXRalpha heterodimers. Strategies to reverse this resistance include the use of the MAP kinase inhibitor, PD098059, and a non-phosphorylatable hRXRalpha mutant, Ala260, which completely abolishes RXR phosphorylation and restores the function of both 1,25(OH)(2)D(3) and a specific RXR ligand, LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl)ethenyl]-benzoic acid). In addition, we show that a vitamin D analog with low calcemic activity (EB1089) is more potent than 1,25(OH)(2)D(3) in inhibiting cancer cell growth in this system. Targeted therapy with selective analogs such as EB1089, in combination with the inhibition of phosphorylation of the RXR, could play a critical role in the development of strategies for cancer treatment.

Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice.

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.