Defects in transforming growth factor-β signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-rasHa transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.

d-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, d-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P4 levels. To define a specific function of Ins(1,4,5,6)P4, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl−) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P4 antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)P4 interfered with the PtdInsP3 effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P4 in Salmonella-infected epithelia may promote Cl− flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP3.

Dendritic cell ontogeny: A human dendritic cell lineage of myeloid origin

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor α chain (IL-3Rαhi). The CD34+IL-3Rαhi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3Rαhi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.

Cytokine-mediated enhancement of epidermal growth factor receptor expression provides an immunological approach to the therapy of pancreatic cancer

The increased expression of epidermal growth factor receptor induced by tumor necrosis factor α renders pancreatic cancer cells more susceptible to antibody-dependent cellular cytotoxicity by a mAb specific for this receptor. Laboratory studies with athymic mice bearing xenografts of human pancreatic cancer cells demonstrated a cytokine-induced ability of the mAb to cause significant tumor regression. In a phase I/II clinical trial, 26 patients with unresectable pancreatic cancer were enrolled into three cohorts receiving variable amounts of the antibody together with a constant amount of tumor necrosis factor α. With increasing doses of antibody, the growth of the primary tumor was significantly inhibited. This was reflected by a longer median survival, with one complete remission lasting for 3 years obtained with the highest dose of antibody employed. Thus, a combination of the cytokine, tumor necrosis factor α, with a mAb to the epidermal growth factor receptor offers a potentially useful approach for the treatment of pancreatic cancer.

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6–4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 × 106 members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7–10 ng/ml (110–160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37°C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Androgen-repressed phenotype in human prostate cancer

An androgen-repressed human prostate cancer cell line, ARCaP, was established and characterized. This cell line was derived from the ascites fluid of a patient with advanced metastatic disease. In contrast to the behavior of androgen-dependent LNCaP and its androgen-independent C4-2 subline, androgen and estrogen suppress the growth of ARCaP cells in a dose-dependent manner in vivo and in vitro. ARCaP is tumorigenic and highly metastatic. It metastasizes to the lymph node, lung, pancreas, liver, kidney, and bone, and forms ascites fluid in athymic hosts. ARCaP cells express low levels of androgen receptor mRNA and prostate-specific antigen mRNA and protein. Immunohistochemical staining shows that ARCaP cells stain intensely for epidermal growth factor receptor, c-erb B2/neu, and c-erb B3. Staining is negative for chromogranin A and positive for bombesin, serotonin, neuron-specific enolase, and the c-met protooncogene (a hepatic growth factor/scatter factor receptor). ARCaP cells also secrete high levels of gelatinase A and B and some stromelysin, which suggests that this cell line may contain markers representing invasive adenocarcinoma with selective neuronendocrine phenotypes. Along with its repression of growth, androgen is also found to repress the expression of prostate-specific antigen in ARCaP cells as detected by a prostate-specific antigen promoter–β-galactosidase reporter assay. Our results suggest that the androgen-repressed state may be central to prostate cancer progression and that advanced prostate cancer can progress from an androgen-independent to an androgen-repressed state.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (−) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER− and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER− and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER− cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER− human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF–EGFR interaction is mediated via activation of nuclear factor κB (NF-κB). The basal level of active NF-κB in ER− cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-κB activation factor. NF-κB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER− cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294–002, blocked this event, suggesting a role of the former in the activation of NF-κB by EGF. Go6976, a well-characterized NF-κB inhibitor, blocked EGF-induced NF-κB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER− cells. Thus Go6976 and similar NF-κB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER− breast cancer patients.

p63 identifies keratinocyte stem cells

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Extracellular matrix composition determines the transcriptional response to epidermal growth factor receptor activation

The transcriptional response to epidermal growth factor (EGF) was examined in a cultured cell model of adhesion. Gene expression was monitored in human embryonic kidney cells (HEK293) after attachment of cells to the extracellular matrix (ECM) proteins, laminin, and fibronectin, by using complementary DNA micorarrays printed with 1,718 individual human genes. Cluster analysis revealed that the influence of EGF on gene expression, either positive or negative, was largely independent of ECM composition. However, clusters of EGF-regulated genes were identified that were diagnostic of the type of ECM proteins to which cells were attached. In these clusters, attachment of cells to a laminin or fibronectin substrata specifically modified the direction of gene expression changes in response to EGF stimulation. For example, in HEK293 cells attached to fibronectin, EGF stimulated an increase in the expression of some genes; however, genes in the same group were nonresponsive or even suppressed in cells attached to laminin. Many of the genes regulated by EGF and ECM proteins in this manner are involved in ECM and cytoskeletal architecture, protein synthesis, and cell cycle control, indicating that cell responses to EGF stimulation can be dramatically affected by ECM composition.

Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was succesfully reconstructed in vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, α-6 and β-1 integrins, was observed in the basement membrane zone, and β-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, the in vitro reconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency of in vitro reconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability of in vitro reconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.