Biomedical subjects

L L Licato

Publications and source records attributed to L L Licato.

8 recordsLinked to original sources

PubMed2001

A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels.

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.

Antigens, CD↗

PubMed2000

Cellular differentiation causes a selective down-regulation of interleukin (IL)-1beta-mediated NF-kappaB activation and IL-8 gene expression in intestinal epithelial cells.

Interleukin (IL)-1beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappaB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1beta-mediated NF-kappaB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1beta-mediated IkappaB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1beta-stimulated HT-29 cells, in contrast to Caco-2 cells. kappaB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1beta signaling pathway inhibiting both NF-kappaB and JNK activity in response to IL-1beta. This relative unresponsiveness to IL-1beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.

Cell Differentiation↗

PubMed2000

Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, Raf-independent mechanism.

BACKGROUND & AIMS: Infectious diarrhea caused by viruses plus enterotoxigenic bacteria is often more severe than diarrhea induced by either pathogen alone. We postulated that the increased cell adenosine 3',5'-cyclic monophosphate (cAMP) concentration observed during infection by enterotoxigenic organisms retards the intestinal repair process by blocking activation of mitogen-activated protein kinases (MAPKs) in proliferating intestinal cells. METHODS: We evaluated the effects of glutamine on MAPK activity, thymidine incorporation, and cell number in glutamine-starved and -sufficient rat intestinal crypt cells (IEC-6). RESULTS: In glutamine-starved cells, 10 mmol/L glutamine in the absence of serum stimulated [(3)H]thymidine incorporation 8-fold. This effect was inhibited by 60% with 8-(4-chlorophenylthio) (8-CPT)-cAMP (100 micromol/L) + isobutyl methylxanthine (100 micromol/L). In cells not starved of glutamine, glutamine stimulated thymidine incorporation by 3-fold, and 8-CPT-cAMP completely blocked the mitogenic effect. Inhibition of proliferation by cAMP persisted for at least 68 hours after cAMP removal. In vitro kinase assays showed that glutamine signaling requires an intact ERK (extracellular signal-related kinase) pathway in unstarved cells. In starved cells, at least one other pathway (JNK) was activated by glutamine, and the mitogenic inhibition by 8-CPT-cAMP was incomplete. Other intestinal fuels (glucose and acetate) were not mitogenic. CONCLUSIONS: Increased levels of intracellular cAMP inhibit ERKs but only partially reduce glutamine-stimulated proliferation in enterocytes adapted to low glutamine.

Cell Division↗

PubMed1999

Multiple interleukin-2 signaling pathways differentially regulated by microgravity.

Defects in innate immunity have been demonstrated in astronauts after space flight. To investigate the role of microgravity on innate immune function, we evaluated NK and LAK activity of human PBMC stimulated with IL-2 under conditions of simulated microgravity, by using a rotating wall vessel (RWV) culture system. Under these conditions, both NK and LAK activity were generated at levels comparable to those found in static flask cultures. The phenotype of the activated PBMC was similar between the two culture conditions, with one notable exception: the IL-2 receptor alpha chain (CD25), which failed to be upregulated in simulated microgravity. To further investigate this change in IL-2 signaling, we examined the ability of IL-2 to induce secondary cytokines. The production of IFNgamma, IL-1beta, and TNFalpha was almost completely abrogated in the microgravity cultures, suggesting that the IL-2 signaling pathways leading to various IL-2-mediated effects are differentially regulated under bioreactor culture conditions.

Cells, Cultured↗

PubMed1998

Analysis of signaling protein kinases in human colon or colorectal carcinomas.

Extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases are highly activated in an in vivo rat model of colorectal carcinogenesis. In addition, other protein kinases such as c-Src and c-Yes have been shown to be up-regulated in some human colon cancers. To evaluate the activity of these kinases in human colorectal carcinomas, we examined colon cancers and adjacent normal intestinal mucosa from 11 patients. Moderate increases in ERK and JNK activities, in addition to up-regulation of c-Src, p125FAK, and tyrosine-phosphorylated proteins, were observed in a subset of the colorectal carcinomas. There was a significant correlation found between levels of c-Src, p125FAK, and tyrosine-phosphorylated proteins, as well as between c-Src protein levels and JNK activity. This is the first report that examines several different kinases as markers to characterize colorectal cancers in the same carcinoma sample, allowing the determination of correlations between markers in the same tumors.

Adenocarcinoma↗

PubMed1998

Mediation by NF-kappa B of cytokine induced expression of intercellular adhesion molecule 1 (ICAM-1) in an intestinal epithelial cell line, a process blocked by proteasome inhibitors.

BACKGROUND/AIMS: The gene promoter for the intercellular adhesion molecule ICAM-1 possesses binding sites for several transcriptional factors, including nuclear factor kappa B (NF-kappa B). The role of NF-kappa B in ICAM-1 gene regulation was therefore examined by using different proteasome inhibitors in tumour necrosis factor alpha (TNF-alpha) stimulated IEC-6 rat intestinal epithelial cells. METHODS: ICAM-1 expression was analysed by enzyme linked immunosorbent assay (ELISA), reverse transcriptase polymerase chain reaction, and immunohistochemistry. Steady state levels of cytoplasmic I kappa B protein were evaluated by western blot, and nuclear translocation of NF-kappa B was determined by electrophoretic mobility shift assay and immunofluorescence staining. Cell adhesion was assayed by measuring the binding of fluorescence labelled MOLT-4 cells. RESULTS: TNF-alpha induced ICAM-1 mRNA and protein expression in IEC-6 cells, which was followed by increased adhesion of MOLT-4 lymphocytes. Blocking TNF-alpha induced I kappa B alpha degradation with proteasome inhibitors reduced TNF-alpha induced NF-kappa B activation and ICAM-1 gene induction and notably decreased MOLT-4 cell adhesion without affecting Jun N-terminal kinase (JNK/SAPK) activity or de novo protein synthesis. CONCLUSION: TNF-alpha induction of ICAM-1 expression is mediated by the transcription factor NF-kappa B and can be inhibited by blocking I kappa B alpha degradation. Thus the I kappa B/NF-kappa B system is a promising target for pharmacological modulation of the expression of adhesion molecules and other inflammatory genes in the intestine.

Animals↗

PubMed1998

Cytokines induce NF-kappaB in activated but not in quiescent rat hepatic stellate cells.

The hepatic stellate cell (HSC), after a fibrogenic stimulus, is transformed from a quiescent to an activated phenotype, including the induction of responsiveness to a variety of agonists. We investigated the activation of nuclear factor-kappaB (NF-kappaB) and the expression of the NF-kappaB-responsive genes intercellular adhesion molecule 1 (ICAM-1) and macrophage inflammatory protein-2 (MIP-2) in freshly isolated and culture-activated HSC by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta. Inhibitor-kappaB was rapidly (<15 min) degraded, and NF-kappaB activity was induced in culture-activated but not in freshly isolated HSC after cytokine stimulation. After 30 min of stimulation, immunofluorescence revealed that the NF-kappaB p65 subunit was predominantly found in the nuclei of activated HSC compared with the cytoplasmic localization in unstimulated cells. No nuclear translocation appeared in freshly isolated HSC after stimulation, despite the presence of functional TNF-alpha receptors. NF-kappaB nuclear translocation appeared first partially after 4-5 days and completely after 9 days in culture. Consistent with this time course TNF-alpha induced the mRNA of the NF-kappaB-dependent genes ICAM-1 and MIP-2 in activated but not in quiescent HSC. Therefore, cytokines induce NF-kappaB activity and ICAM-1 and MIP-2 mRNAs in activated but not in quiescent HSC, through a postreceptor mechanism of regulation.

Animals↗

PubMed1997

In vivo activation of mitogen-activated protein kinases in rat intestinal neoplasia.

BACKGROUND & AIMS: To investigate whether mitogen-activated protein kinase (MAPK) cascades might play a role in the progression of colon cancer, c-Jun N-terminal kinase (JNK) and extracellular signal regulating kinase (ERK) activity during colonic tumorigenesis were examined. METHODS: The 1,2-dimethylhydrazine (DMH)-induced colon carcinoma model was used to study the activation of these kinases during intestinal carcinogenesis. Male Sprague-Dawley rats were injected with DMH for 24 weeks. Normal-appearing intestinal mucosa from control and treated animals and DMH-induced intestinal tumors were assayed for JNK and ERK activity using solid phase in vitro kinase assays. Tumors were typed for mutations in the K-ras gene. RESULTS: There was little or no difference in JNK and ERK activity in hyperproliferative mucosa from DMH-treated animals compared with normal mucosa from control animals. However, in 16 colonic neoplasms, an average of 23-fold and 29-fold increases in JNK and ERK activities were observed, respectively, over control levels. In addition, activating protein-1 binding was strongly induced in the colonic tumors. Activation did not correlate with the presence of mutations in K-ras. CONCLUSIONS: Both the JNK and ERK MAPKs are highly activated during late progression of colorectal carcinoma. This change is dependent on the tumorigenic state rather than changes in proliferation.

1,2-Dimethylhydrazine↗