The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice.

BACKGROUND & AIMS: Keratins are intermediate filaments that are critical in cytoskeletal organization. Their roles in cellular processes are underscored by inherited human diseases in which germline mutations of keratins are found, as well as by transgenic and knockout mouse models that recapitulate those diseases. Keratin 19 (K19) has unique structural properties and developmental and spatial expression patterns. This suggests that K19 expression may correlate with important cell fate decisions in gastrointestinal tract epithelia. METHODS: We used mouse K19 5' untranslated region and promoter sequences and fused it to the lacZ reporter gene in a transgene construct. Characterization was by beta-galactosidase expression and X-gal histochemistry in gastrointestinal epithelia. Because endogenous K19 protein is transcriptionally regulated by the Kruppel-like transcription factor 4 (KLF4), we determined the spatial expression patterns of KLF4 and K19 in relationship to the lacZ reporter gene product. RESULTS: K19-lacZ transgenic mice were found to have reporter gene expression in an epithelial-specific pattern. Expression was restricted to ductal epithelial cells in the pancreas, surface colonocytes, small intestinal villi, and gastric isthmus cells. Transgene expression correlated with K19 and KLF4 protein expression in the pancreas and stomach and was overlapping in the small and large intestine. CONCLUSIONS: The K19 promoter may be a useful tool to study epithelial cell biology and subsequent transdifferentiation programs, particularly the pancreas and stomach.

Activation of protein kinase Calpha inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest.

We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)alpha. PKCalpha subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKC(&agr;) activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCalpha, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKC(&agr;) activation arrested cells in the G(1) phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21(cip1) cyclin-dependent kinase inhibitors. Levels of p21(cip1) remained elevated and CDK2 activity repressed in spite of PKCalpha downregulation, indicating that downstream effectors of PKCalpha are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCalpha-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCalpha results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.

The tissue-dependent keratin 19 gene transcription is regulated by GKLF/KLF4 and Sp1.

Keratins play critical roles in cellular differentiation and cytoskeletal organization. Keratin 19 (K19) is unique because it has been implicated as a marker of stem cells in some tissues, such as the hair follicle in the skin. It is also associated with malignant transformation in esophageal and pancreatic cancers. Here, we show that the K19 promoter is active in a subset of gastrointestinal cancer cells derived from esophageal and pancreas but inactive in other contexts. This activity was mapped to a short region containing an overlapping binding site for gut-enriched Krüppel-like factor (GKLF/KLF4) and Sp1. GKLF has a higher binding affinity and is the predominant binding factor in cells with low Sp-1 protein levels. Pancreatic acinar cells normally do not express K19, but overexpression of GKLF and Sp1 in these cells leads to aberrant expression, similar to what is observed in pancreatic cancer. These results demonstrate the functional interaction of ubiquitous and tissue-restricted transcription factors in determining tissue- and neoplasm-specific patterns of gene expression.

Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

Molecular mechanism of interferon alfa-mediated growth inhibition in human neuroendocrine tumor cells.

BACKGROUND & AIMS: Although human neuroendocrine tumors respond to interferon (IFN)-alpha treatment in vivo, the underlying mechanisms of growth inhibition are poorly understood. To characterize the antiproliferative effects at a molecular level, we explored the growth-regulatory action of IFN-alpha in the human neuroendocrine tumor cell lines BON and QGP1. METHODS: IFN-alpha receptor expression and signal transduction were examined by reverse-transcription polymerase chain reaction, immunoblotting, subcellular fractionation, and transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. Expression and activity of cell cycle-regulatory molecules were determined by immunoblotting and histone H1-kinase assays. RESULTS: Both cell lines expressed IFN-alpha receptor mRNA transcripts. Ligand binding initiated phosphorylation of Jak kinases and Stat transcription factors, resulting in Stat activation, nuclear translocation, and transcription from an ISRE-reporter construct. Prolonged IFN-alpha treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-alpha-treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G(1) synchronized QGP1 cells. IFN-alpha-treated cells entered S phase in parallel to control cultures, but their progress into G(2)/M phase was delayed. Both cellular cyclin B levels and CDC 2 activity were substantially reduced. The extent and time course of this reduction corresponded to the observed S-phase accumulation. CONCLUSIONS: IFN-alpha directly inhibits growth of human neuroendocrine tumor cells by specifically delaying progression through S phase and into G(2)/M. These cell cycle changes are associated with inhibition of cyclin B expression, resulting in reduced CDC2 activity.

All-trans-retinoic acid-mediated growth inhibition involves inhibition of human kinesin-related protein HsEg5.

In this study we used differential display reverse transcription-polymerase chain reaction to search for differentially expressed all-trans-retinoic acid (ATRA)-responsive genes in pancreatic carcinoma cells. We identified the kinesin-related protein HsEg5, which plays an essential role in spindle assembly and spindle function during mitosis, as a novel molecule involved in ATRA-mediated growth inhibition. Using Northern and Western blot analysis we demonstrated that ATRA significantly inhibits HsEg5 expression in various pancreatic carcinoma cell lines as well as in HaCat keratinocytes. Inhibition of HsEg5 expression by ATRA occurs at the posttranscriptional level. As a consequence, tumor cells synchronized in S-phase revealed a retarded progression through G2/M phase of the cell cycle indicating that HsEg5 inhibition results in a delayed progression through mitosis. Furthermore, a significant decrease of HsEg5 protein expression achieved by antisense transfection revealed a significant growth inhibition compared with control cells. Therefore, HsEg5 represents a novel molecule involved in ATRA-mediated growth inhibition, suggesting that vitamin A derivatives can interact with the bipolar spindle apparatus during mitosis.

Fra-1: a novel target for retinoid action.

Using a cDNA gene expression array system, we identified fra-1 as a novel target molecule for retinoid action in the human pancreatic carcinoma cell line Dan-G. Retinoid treatment resulted in a significant and time-dependent induction of the fra-1 expression on the post-transcriptional level. Supershift assays revealed that fra-1 participates in the activator protein 1 complex together with c-fos, c-jun and junB. Transient transfection experiments using a reporter plasmid containing an activator protein site upstream of the CAT reporter gene revealed that the phorbolester-induced CAT activity was suppressed by retinoids. Since fra-1 lacks a transactivation function, we therefore suggest that the retinoid-mediated induction of fra-1 might function as a negative regulator of the activator protein 1 activity in human pancreatic carcinoma cells.

A phase II pilot trial of 13-cis retinoic acid and interferon-alpha in patients with advanced pancreatic carcinoma.

BACKGROUND: Advanced unresectable pancreatic adenocarcinoma has a dismal prognosis. The authors previously have shown that retinoic acid (RA) and interferon-alpha (IFN-alpha) inhibit growth and induce differentiation in human pancreatic carcinoma cells in vitro and in vivo. The purpose of this trial was to examine the feasibility and tolerability of a combination therapy of 13-cis RA and IFN-alpha in patients with advanced unresectable pancreatic carcinoma. METHODS: Twenty-two patients (median age, 62 years) with histologically confirmed, unresectable pancreatic adenocarcinoma classified as International Union Against Cancer Stage III (5 patients) or IV (17 patients) were included. Patients received 1 mg/kg body weight 13-cis RA orally and 6 million IU IFN-alpha subcutaneously daily. Restaging by ultrasound, computed tomography scan, and chest X-ray was performed every 2 months. RESULTS: No complete remission and 1 partial remission (PR) (4.5%) were observed. Fourteen patients (63.6%) demonstrated stable disease with a median duration of 5.0 months (range, 2.3-17.7+ months). Toxicity mainly was related to IFN-alpha and predominantly was hematologic (no toxicity was World Health Organization [WHO] Grade 4 and 13.6% were WHO Grade 3). Nonhematologic toxicities did not exceed Grade 2 (skin and oral mucosa) and mainly were related to 13-cis RA. The median survival of the patients with Stage III disease was 8.7 months (range, 6.8-23.9+ months) and was 7.4 months for patients with Stage IV disease (range, 0.9-19.2+ months), resulting in a median overall survival of 7.7 months (range, 0.9-23.9+ months). CONCLUSIONS: Combination therapy with 13-cis RA and IFN-alpha is feasible and well tolerated in patients with advanced pancreatic carcinoma. Based on the median survival rates observed in this study this combination should be investigated further in Phase III trials.

Retinoic acid receptor alpha mediates growth inhibition by retinoids in rat pancreatic carcinoma DSL-6A/C1 cells.

During carcinogenesis, pancreatic acinar cells can dedifferentiate into ductal adenocarcinoma of the pancreas. DSL-6A/C1 cells represent an in vitro model of this carcinogenic sequence. This study was designed to examine the effects of retinoids on cell growth in DSL-6A/C1 cells and to characterize further the molecular mechanisms underlying the antiproliferative actions of retinoids. Treatment of DSL-6A/C1 cells with retinoids results in a time- and dose-dependent inhibition of cell growth, paralleled by a retinoid-mediated transactivation of a pTK::betaRAREx2-luciferase reporter construct transiently transfected into DSL-6A/C1 cells. Retinoid receptor expression was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) using subtype-specific primers and demonstrated expression of retinoic acid receptor alpha (RAR-alpha), RAR-beta and retinoid X receptor alpha (RXR-alpha). Using a panel of receptor subtype-specific agonists, the RAR-alpha specific agonist Ro 40-6055 was the most potent retinoid in terms of growth inhibition. Furthermore, all-trans-retinoic acid-mediated growth inhibition and transactivation was completely blocked by the RAR-alpha-specific antagonist Ro 41-5253. In summary, the RAR-alpha subtype predominantly mediates the antiproliferative effects of retinoids in DSL-6A/C1 cells. Furthermore, this cell system provides a feasible tool to study the molecular mechanisms underlying the growth inhibitory effects of retinoids in ductal pancreatic carcinoma cells derived from a primary acinar cell phenotype.