Study of retinoic acid signaling in cancer cells: Cloning and characterization of retinoic acid responsive genes

Retinoids such as all-trans-retinoic acid (ATRA) are promising agents for cancer chemoprevention and therapy. ATRA can cause growth inhibition, induction of differentiation and apoptosis of a variety of cancer cells. These effects are thought to be mediated by nuclear retinoids receptors which are involved in ligand-dependent transcriptional activation of downstream target genes. Using differential display, we identified several retinoic acid responsive genes in the head and neck squamous carcinoma cells and lung cancer cells, including tissue type transglutaminase, cytochrome P450-related retinoic acid hydroxylase, and a novel gene, designated RAIG1. RAIG1 has two transcripts of 2.4 and 6.8 kbp, respectively, that are generated by alternative selection of polyadenylation sites. Both transcripts have the same open reading frame that encodes a protein comprised of 357 amino acid residues. The deduced RAIG1 protein sequence contains seven transmembrane domains, a signature structure of G protein-coupled receptors. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid and dose-dependent. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. The locus for RAIG1 gene was mapped to a region between D12S358 and D12S847 on chromosome 12p12.3-p13. Our study of the novel retinoic acid induced gene RAIG1 provide evidence for a possible interaction between retinoid and G protein signaling pathways.^ We further examined RAIG1 expression pattern in a panel of 84 cancer cell lines of different origin. The expression level varies greatly from very high to non-detectable. We selected a panel of different cancer cells to study the effects of retinoids and other differentiation agents. We observed: (1) In most cases, retinoids (including all-trans retinoic acid, 4HPR, CD437) could induce the expression of RAIG-1 in cells from cancers of the breast, colon, head and neck, lung, ovarian and prostate. (2) Compare to retinoids, butyrate is often a more potent inducer of RAIG-1 expression in many cancer cells. (3) Butyrate, Phenylacetate butyrate, (R)P-Butyrate and (S)P-Butyrate have different impact on RAIG1 expression which varies among different cell lines. Our results indicate that retinoids could restore RAIG1 expression that is down-regulated in many cancer cells.^ A mouse homologous gene, mRAIG1, was cloned by 5$\sp\prime$ RACE reaction. mRAIG1 cDNA has 2105 bp and shares 63% identity with RAIG1 cDNA. mRAIG1 encodes a polypeptide of 356 amino acid which is 76% identity with RAIG1 protein. mRAIG1 protein also has seven transmembrane domains which are structurally identical to those of RAIG1 protein. Only one 2.2 kbp mRAIG1 transcript could be detected. The mRAIG1 mRNA is also highly expressed in lung tissue. The expression of mRAIG1 gene could be induced by ATRA in several mouse embryonal carcinoma cells. The induction of mRAIG1 expression is associated with retinoic acid-induced neuroectoderm differentiation of P19 cells. Similarity in cDNA and protein sequence, secondary structure, tissue distribution and inducible expression by retinoic acid strongly suggest that the mouse gene is the homologue of the human RAIG1 gene. ^

PHENOTYPIC ASSOCIATION OF GENE AMPLIFICATION WITH MULTIPLE DRUG RESISTANCE IN VINCRISTINE RESISTANT CHINESE HAMSTER OVARY CELLS

Resistance of tumors to pharmacologic agents poses a significant problem in the treatment of human malignancies. This study overviews the scope of clinical resistance and focuses upon current research attempts toward investigation of the phenomenon of multidrug resistance (MDR).^ The objective of this investigation was to determine whether gene amplification had a role in the development of the MDR phenotype in Chinese hamster ovary cells (CHO) primarily selected for resistance to vincristine (VCR). A DNA fragment, previously shown to be amplified in two independently derived Chinese hamster cell lines exhibiting the MDR phenotype, was also amplified in VCR hamster lines. Sequences flanking this fragment were shown to contain coding information for a 4.3 kb transcript overproduced in VCR cells. These sequences were not enriched in double minute DNA preparations isolated from VCR cells. There was an approximately forty-fold increase in both the level of gene amplification and transcript overproduction in the VCR cell lines, independent of the level of primary resistance. This DNA amplification and overproduction of the 4.3 kb transcript was also demonstrated in CHO cells independently selected for resistance to Adriamycin and vinblastine.^ All the DNA sequences of two hamster cDNA clones containing 785 and 932 base pair inserts showed direct homology to the published mouse mdr sequences (about 90%). This sequence conservation held for only portions of the gene when the human mdr1 sequences were compared with those from either the mouse or hamster.^ Somatic cell hybrids, constructed between VCR CHO cells and sensitive murine cells, were used to determine whether there was a functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of MDR-associated amplified sequences, overexpression of the mRNA encoded by these sequences, overexpression of the mRNA encoded by these sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the VCR CHO cells which is responsible for MDR in these cells. ^

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems

In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Constitutive notch signaling in adult transgenic mice inhibits bFGF-induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock-out and transgenic mice have precluded studies of the role of Notch post-natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre-conditional expression of the constitutively active intracellular domain of Notch1 (IC-Notch1). Double transgenic IC-Notch1/Tie2-Cre embryos with endothelial specific IC-Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline-inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC-Notch1 expression inhibited bFGF-induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF-induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies.

Monitoring and Switching of First-line Antiretroviral Therapy in sub-Saharan Africa: Collaborative Analysis of Adult Treatment Cohorts.

BACKGROUND HIV-1 viral load (VL) testing is recommended to monitor antiretroviral therapy (ART) but not universally available. We examined monitoring of first-line and switching to second-line ART in sub-Saharan Africa, 2004-2013. METHODS Adult HIV-1 infected patients starting combination ART in 16 countries were included. Switching was defined as a change from a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen to a protease inhibitor (PI)-based regimen, with a change of ≥1 NRTI. Virological and immunological failures were defined per World Health Organization criteria. We calculated cumulative probabilities of switching and hazard ratios with 95% confidence intervals (CI) comparing routine VL monitoring, targeted VL monitoring, CD4 cell monitoring and clinical monitoring, adjusted for programme and individual characteristics. FINDINGS Of 297,825 eligible patients, 10,352 patients (3·5%) switched during 782,412 person-years of follow-up. Compared to CD4 monitoring hazard ratios for switching were 3·15 (95% CI 2·92-3·40) for routine VL, 1·21 (1·13-1·30) for targeted VL and 0·49 (0·43-0·56) for clinical monitoring. Overall 58.0% of patients with confirmed virological and 19·3% of patients with confirmed immunological failure switched within 2 years. Among patients who switched the percentage with evidence of treatment failure based on a single CD4 or VL measurement ranged from 32·1% with clinical to 84.3% with targeted VL monitoring. Median CD4 counts at switching were 215 cells/µl under routine VL monitoring but lower with other monitoring (114-133 cells/µl). INTERPRETATION Overall few patients switched to second-line ART and switching occurred late in the absence of routine viral load monitoring. Switching was more common and occurred earlier with targeted or routine viral load testing.

Psychological distress in medical seeking ED care for somatic reasons: results of a systematic review

Objectives: The aim of this systematic literature review is to investigate (A) currently used instruments for assessing psychological distress, (B) the prevalence of psychological distress in medical emergency department (ED) patients with acute somatic conditions and (C) empirical evidence on how predictors are associated with psychological distress. Methods: We conducted an electronic literature search using three databases to identify studies that used validated instruments for detection of psychological distress in adult patients presented to the ED with somatic (non-psychiatric) complaints. From a total of 1688 potential articles, 18 studies were selected for in-depth review. Results: A total of 13 instruments have been applied for assessment of distress including screening questionnaires and briefly structured clinical interviews. Using these instruments, the prevalence of psychological distress detected in medical ED patients was between 4% and 47%. Psychological distress in general and particularly depression and anxiety have been found to be associated with demographic factors (eg, female gender, middle age) and illness-related variables (eg, urgency of triage category). Some studies reported that coexisting psychological distress of medical patients identified in the ED was associated with physical and psychological health status after ED discharge. Importantly, during routine clinical care, only few patients with psychological distress were diagnosed by their treating physicians. Conclusions: There is strong evidence that psychological distress is an important and prevalent cofactor in medically ill patients presenting to the ED with harmful associations with (subjective) health outcomes. To prove causality, future research should investigate whether screening and lowering psychological distress with specific interventions would result in better patient outcomes.

Mesenchymal Stem Cells from Wharton's Jelly and Amniotic Fluid

The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Wharton's jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BMMSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJMSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

Adult Asylum Seekers from the Middle East Including Syria in Central Europe: What Are Their Health Care Problems?

Background Forced displacement related to persecution and violent conflict has reached a new peak in recent years. The primary aim of this study is to provide an initial overview of the acute and chronic health care problems of asylum seekers from the Middle East, with special emphasis on asylum seekers from Syria. Methods Our retrospective data analysis comprised adult patients presenting to our emergency department between 01.11.2011 and 30.06.2014 with the official resident status of an “asylum seeker” or “refugee” from the Middle East. Results In total, 880 patients were included in the study. Of these, 625 (71.0%) were male and 255 (29.0%) female. The median age was 34 (range 16–84). 222 (25.2%) of our patients were from Syria. The most common reason for presentation was surgical (381, 43.3%), followed by medical (321, 36.5%) and psychiatric (137, 15.6%). In patients with surgical presentations, trauma-related problems were most common (n = 196, 50.6%). Within the group of patients with medical presentation, acute infectious diseases were most common (n = 141, 43.9%), followed by neurological problems (n = 70, 21.8%) and gastrointestinal problems (n = 47, 14.6%). There were no differences between Syrian and non-Syrian refugees concerning surgical or medical admissions. The most common chronic disorder of unclear significance was chronic gastrointestinal problems (n = 132, 15%), followed by chronic musculoskeletal problems (n = 108, 12.3%) and chronic headaches (n = 78, 8.9%). Patients from Syria were significantly younger and more often suffered from a post-traumatic stress disorder than patients of other nationalities (p<0.0001, and p = 0.05, respectively). Conclusion Overall a remarkable number of our very young group of patients suffered from psychiatric disorders and unspecified somatic symptoms. Asylum seekers should be carefully evaluated when presenting to a medical facility and physicians should be aware of the high incidence of unspecified somatic symptoms in this patient population.In general, there is no major difference between asylum seekers from Syria when compared to other nationalities of asylum seekers from the Middle East.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration.

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^

CHARACTERIZATION OF HIGH MOBILITY GROUP AND HISTONE H1 PROTEIN LEVELS AND SYNTHESIS DURING SPERMATOGENESIS IN THE RAT

In order to propose a role for internucleosomal high mobility group proteins (HMGs), and HI histone variants study of their levels and synthesis in a system of development and differentiation--rat spermatogenesis--was undertaken. HMG1, 2, 14, and 17 were isolated from rat testes and found to be very similar to calf thymus HMGs. Testis levels of HMGs, relative to DNA, were equivalent to other rat tissues for HMG1 (13 ug/mg DNA), HMG14 (2 ug/mg DNA), and HMG17 (5 ug/mg DNA). HMG2 levels were different among rat tissues, with three groups observed: (1) nonproliferating tissues (1-5 ug/mg DNA); (2) proliferating tissues (8-13 ug/mg DNA); and (3) the testis (32 ug/mg DNA). Other species (toad, opposum, mouse, dog, and monkey) showed the same testis-specific increase of HMG2. Populations of purified testis cell types were separated by centrifugal elutriation and density gradient centrifugation from adult and immature rat testes. Pachytene spermatocytes and early spermatids (56 and 47 ug/mg DNA, respectively) caused the testis-specific increase of HMG2 levels. Cell types preceding pachytenes (types A and B spermatogonia, mixtures of spermatogonia and early primary spermatocytes, and early pachytenes contained HMG2 levels similar to proliferating tissues (12 ug/mg DNA). Late spermatids did not contain HMGs. Somatic Sertoli and Leydig cells (2 ug/mg DNA) exhibited HMG2 levels similar to nonproliferating tissues. HMGs synthesized in spermatogonia and spermatocytes had similar specific activities, but early spermatids did not synthesize HMGs. Germ cells also contained an HMG2 species (on acid-urea gels) not found in somatic tissues. Other investigators have shown that HMGs may be associated with transcriptional or replicative processes. Thus, it is proposed that HMG2 plays a role in modulatable gene expression, while HMG1 is associated with housekeeping functions.^ HI histone variants were also studied throughout spermatogenesis. The minor somatic variant, HIa, is the predominant variant in spermatogonia and early primary spermatocytes. In early pachytenes, the testis-specific variant, HIt, is first synthesized and appears, largely replacing somatic variants HIbcd and e by late pachytene stage. Early spermatids contain the same HI composition as pachytenes, but do not synthesize HI histones. HI('0) is present in low amounts in all germ cells. These results suggest that expression of HI variants is developmentally controlled.^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^