Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers.

Exposure of LLC-PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C-alpha (PKC-alpha) from cytosolic to membrane-associated compartments. With a very similar time course, a dramatic and sustained increase in tight junctional (paracellular) permeability occurs. This increased permeability extends not only to salts and sugars but macromolecules as well.

The smooth muscle cell membrane during atherogenesis: a potential target for amlodipine in atheroprotection.

Background: Atherosclerotic disease has been present in the human population apparently from the beginning of time. However, it has only been in the 20th century that improvements in the control of infectious diseases have allowed the average life span to increase to the point where atherosclerosis has been able to affect the general population. By the middle of the 20th century, atherosclerosis had reached epidemic levels, and it is currently pandemic and increasing worldwide.

Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer.

Breast cancers often show reduced expression of the transmembrane cell-cell adhesion protein, E-cadherin. In addition, approximately half of breast carcinomas express P-cadherin, which correlates with poor survival. A large fragment of the E-cadherin extracellular domain can be detected in serum, and it has been proposed that an increase in serum E-cadherin can denote the presence of a tumor.

Regulation of protein kinase C-delta and -epsilon isoforms by phorbol ester treatment of LLC-PK1 renal epithelia.

Background: LLC-PK1 renal epithelia are a widely used model for proximal tubular physiology and differentiation. Protein kinase C (PKC) has been observed to play a role in both processes. This study examines the subcellular distribution and down-regulation of PKC-delta and PKC-epsilon isoforms in phorbol ester-treated LLC-PK1 epithelia.

Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(-7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(-7) M TPA for up to 2 hours.

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation.

Wnt-1 dependent activation of the survival factor NF-kappaB in PC12 cells.

Expression of the Wnt-1 oncogene in PC12 cells induces morphological and biochemical changes, including up-regulation of cell adhesion and lack of differentiation in response to growth factors. The survival of PC12 cells is known to be mediated in part by phosphatidylinositol-3 kinase (PI-3 kinase)-dependent activation of the transcription factor nuclear factor-kappaB (NF-kappaB). We investigated the effect of Wnt-1 expression on cell survival and NF-kappaB activation using PC12 cells expressing Wnt-1 (PC12/Wnt1) and a reporter vector in which firefly luciferase expression is under the control of NF-kappaB consensus sequences.

Modification of tight junction function by protein kinase C isoforms.

The regulation of tight junction permeability by a variety of signal transduction pathways is summarized. An emphasis is placed on regulation of paracellular permeability by the protein kinase C family of isoforms, which involves the reporting of a large number of studies using the phorbol ester family of protein kinase C activators. The ability of protein kinase C activation to open epithelial barriers to a very wide range of solutes is emphasized, but then countered with discussion of the role of phorbol esters and protein kinase C activation in epithelial carcinogenesis.

Insulin-like growth factor I-mediated degradation of insulin receptor substrate-1 is inhibited by epidermal growth factor in prostate epithelial cells.

We have sought to determine whether insulin-like growth factor I (IGF-I) regulates the levels of insulin receptor substrate-1 (IRS-1) in prostate epithelial cells. Exposure of prostate epithelial cells to IGF-I in the absence of other growth factors leads to a reduction in IRS-1 levels. Ubiquitin content of IRS-1 is increased in the presence of IGF-I, and inhibitors of the proteasome prevented the reduction of IRS-1 levels seen following IGF-I exposure.

High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation.

Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation. Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis. Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones.

Upregulation of myogenin by N-cadherin adhesion in three-dimensional cultures of skeletal myogenic BHK cells.

Cells of the baby hamster kidney (BHK) line express the skeletal muscle determining transcription factor MyoD but fail to differentiate. Unlike most skeletal myogenic cells, which express multiple members of the cadherin family of cell-cell adhesion proteins, the BHK cells lack a robust cadherin adhesion system. We previously published that forced expression of N- (or E)-cadherin in BHK cells increases the level of endogenous catenins, mediates strong cell-cell adhesion, and enhances differentiation of BHK cells induced to differentiate by placing them in three-dimensional (3-D) culture (Redfield et al.

Oxidative stress and gene regulation.

Reactive oxygen species are produced by all aerobic cells and are widely believed to play a pivotal role in aging as well as a number of degenerative diseases. The consequences of the generation of oxidants in cells does not appear to be limited to promotion of deleterious effects. Alterations in oxidative metabolism have long been known to occur during differentiation and development.

Evidence that the epidermal targets of carcinogen action are found in the interfollicular epidermis of infundibulum as well as in the hair follicles.

Actively cycling, transit-amplifying cells and quiescent cells including stem cells are found in the layer of the epidermis and hair follicles. To determine the origin of skin tumors, we completely removed the interfollicular epidermis of carcinogen-initiated mice by an abrasion technique known to leave the hair follicles undisturbed. The interfollicular epidermis of the abraded mice quickly regenerated from cells in the hair follicles, after which time tumor promotion was begun.

Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin.

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates.

P-cadherin expression in breast carcinoma indicates poor survival.

Background: The cadherin family of cell-cell adhesion molecules and their associated proteins, the catenins, are essential to embryonic development and the maintenance of adult tissues. During development, the homotypic interaction of a particular cadherin with an identical cadherin expressed on a neighboring cell results in the sorting of cells to form distinctive tissues. Cadherins are believed to be tumor suppressors, and their altered expression and function have been associated with tumor development.

Conversion of C57Bl/6 mice from a tumor promotion-resistant to a -sensitive phenotype by enhanced ornithine decarboxylase expression.

A transgenic mouse model was developed in which ornithine decarboxylase (ODC) can be overexpressed in a tissue-specific and regulated manner. Hair follicle keratinocytes were targeted by use of a bovine keratin 6 (K6) promoter/regulatory region, and regulation was accomplished by using the tetracycline-regulated transactivator/tetracycline-response element system. Double-transgenic mice carrying both transgenes (K6/tetracycline-regulatable transactivator protein (tTA) and tetracycline-response element/Odc) on a C57Bl/6 background had no obvious phenotypic abnormalities in the absence (Odc transgene-expressed) of doxycycline (a tetracycline analog) in the drinking water.

Increased tight junctional permeability is associated with the development of colon cancer.

Epithelial tissues act as barriers between two fluid compartments, and the epithelial barrier function is provided by the epithelial cells and the tight junctions (TJs) that connect them. We have shown previously that chronic treatment of a cultured epithelial monolayer with phorbol ester tumor promoters induces an increase in transepithelial paracellular permeability and produces tumor-like polyps, suggesting an association between TJ permeability and tumor formation. In this study, we analyzed the association between TJ permeability and formation of tumors in vivo.

Highly persistent label-retaining cells in the hair follicles of mice and their fate following induction of anagen.

We have identified some unusually persistent label-retaining cells in the hair follicles of mice, and have investigated their role in hair growth. Three-dimensional reconstruction of dorsal underfur follicles from serial sections made 14 mo after complete labeling of epidermis and hair follicles in neonatal mice disclosed the presence of highly persistent label-retaining cells associated with the first-generation follicle involved in the production of the first wave of hairs, commonly called the bulge. The label-retaining cells were most often found on the ventral surface of the first-generation follicle, five cell positions from the base, near the attachment site of the arrector pilorum muscle.

Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis.

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments.

Absence of DNA ligase IV protein in XR-1 cells: evidence for stabilization by XRCC4.

XR-1 is a CHO mutant cell line defective in double strand break repair and V(D)J recombination. These defects are due to a deletion of the XRCC4 gene which encodes a 38-kDa nuclear phosphoprotein. Recent studies have shown that XRCC4 interacts with and enhances the activity of DNA ligase IV in vitro.

Time versus replication dependence of EMS-induced delayed mutation in Chinese hamster cells.

We have previously observed in Chinese hamster cells that ethyl methane sulfonate (EMS) induces mutations which are distributed over at least 10-14 cell divisions following treatment. This delayed appearance of mutations could be explained by EMS-induced lesions which remain in DNA and have a probability that is significantly less than 1.0 of producing base mispairing errors during successive replication cycles (replication-dependent).

EMS and UV-light-induced colony sectoring and delayed mutation in Chinese hamster cells.

Purpose: To review studies of mutagen-induced colony sectoring which demonstrate that UV light and EMS produce delayed mutational events in Chinese hamster ovary cells.

Methods And Results: Since the late 1940s, it has been known that the treatment of a single bacterial or yeast cell with mutagenic agents produces complete mutant colonies (pures) and colonies composed of both mutant and non-mutant cell types (mosaics) with various sectored patterns. A similar sectoring phenomenon has been observed in Chinese hamster ovary cells (CHO) using the DNA alkylating agent ethyl methane sulphonate (EMS) or ultraviolet light.

Cell-specific ecdysone-inducible expression of FLP recombinase in mammalian cells.

The ability of site-specific recombinases, like FLP and Cre, to catalyze alterations in genomic DNA is well established, whereas their application to genetic engineering strategies has been restricted because of the inability to temporally regulate their expression and subsequent recombination events in specific populations of cells. We describe a regulatory system for ecdysone-controlled expression of FLP recombinase. Furthermore, we demonstrate that ecdysone-induced, FLP-mediated site-specific recombination events can be targeted to specific cells.

A composite CMV-IE enhancer/beta-actin promoter is ubiquitously expressed in mouse cutaneous epithelium.

In most existing transgenic mouse models developed for the study of specific genes in the skin, the goal has been to target transgene expression to defined populations of cells in the cutaneous epithelium. Keratin promoters have been especially useful for this purpose. In some instances, however, it may be desirable to express a transgene in all the cells of the cutaneous epithelium.