Immunotargeting and eradication of orthotopic melanoma using a chemokine-enhanced DNA vaccine.

DNA vaccines are attractive candidates for tumor immunotherapy. However, the potential of DNA vaccines in treating established malignant lesions has yet to be demonstrated. Here we demonstrate that transient alteration of either intratumoral or intradermal (ID) chemotactic gradients provide a favorable milieu for DNA vaccine-mediated activation of tumor-specific immune response in both prophylactic and therapeutic settings. Specifically, we show that priming of established B16 ID melanoma lesions via forced intratumoral expression of CCL21 boosted DNA vaccination-dependent systemic cytotoxic immune response leading to the regression of tumor nodules. In this setting, application of CCL20 was not effective likely due to the engagement of the regulatory T cells. However, priming of the skin at DNA vaccine administration sites outside the tumor bed with both CCL20 and CCL21 chemokines along with structural modifications of the DNA vaccine significantly improved vaccine efficacy. This optimized ID vaccination regimen led to the inhibition of distant established melanomas and prolonged tumor-free survival of mice observed in 60% of vaccinated animals with complete tumor remission in 30%. These effects were mediated by extranodal priming and activation of T cells at vaccine administration sites and progressive accumulation of systemic antigen-specific cytotoxic T cells (CTLs) on successive vaccinations. These results underscore the potential of chemokine-enhanced DNA vaccination to mount therapeutic immune response against established tumors.

Zebrafish: an emerging model system for human disease and drug discovery.

In vivo studies represent an essential step in drug development and currently rely largely on mice, yet limitations of mammalian models motivated the search for complementary vertebrate model systems. This review focuses on zebrafish, Danio rerio, as a facile model system to study human disease and drug responses. Zebrafish are particularly suited for this purpose because they represent a vertebrate species, their genome is sequenced, and a large number of synchronously developing, transparent embryos can be produced. Zebrafish embryos are permeable to drugs and can easily be manipulated using well-established genetic and molecular approaches. Here, we summarize recent work on drug discovery and toxicity in zebrafish embryos. In addition, we provide a synopsis of current efforts to establish disease models in zebrafish focusing on neoplasia. The results of these studies highlight the potential of zebrafish as a viable addition to established animal models by offering medium and, potentially, high throughput capabilities.

Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway.

Normal epithelial cells undergo apoptosis when they are denied contact with the extracellular matrix, in a process termed "anoikis." Conversely, malignant epithelial cells typically acquire anchorage independence, i.e., the capacity to survive and grow in the absence of matrix interaction. Here we asked the question whether anoikis is affected by signaling through the EGF receptor (EGFR). We focused on the EGFR because EGFR signaling is frequently deregulated in malignant epithelial cells. We demonstrate that EGFR activation markedly alleviated the requirement of matrix engagement for survival of primary and immortalized human keratinocytes in suspension culture. Protection of epithelial cells through EGFR activation against anoikis was associated with and required sustained MAPK phosphorylation during the early phase of suspension culture. Interestingly, high levels of MAPK phosphorylation were not only required for EGFR-mediated protection against anoikis but also occurred as a consequence of caspase activation at later stages of suspension culture. These results demonstrate that EGFR activation contributes to anchorage-independent epithelial cell survival and identify MAPK activation as an important mechanism in this process.

Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.

The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

Inhibition of metastases of a human melanoma xenograft by monoclonal antibody to the GD2/GD3 gangliosides.

A human melanoma variant cell line was obtained from a lung metastasis that arose spontaneously after we inoculated melanoma cells sc into a nude mouse. In this model, IgG2a monoclonal antibody (MAb) ME 36.1 defining the GD2/GD3 gangliosides inhibited melanoma growth at the primary site and metastatic spread of the cells, whereas an IgG1 variant of MAb ME 36.1 inhibited lung metastasis formation only. Possible mechanisms of antitumor effects of MAb ME 36.1 are discussed.

Independent regulation of growth and SMAD-mediated transcription by transforming growth factor beta in human melanoma cells.

Increased production of transforming growth factor beta (TGF-beta) coupled with resistance to the growth-inhibitory effects of TGF-beta is characteristic of several types of neoplasia including human melanoma. In select epithelial malignancies, lack of TGF-beta-induced growth inhibition is associated with disruptions of TGF-beta-dependent SMAD signaling and transcription. In contrast, the results of the present study indicate intact SMAD-dependent transcription in human melanoma cells, regardless of their proliferative response to exogenous TGF-beta. Furthermore, in some melanoma cell lines constitutive SMAD-dependent transcription was observed, which was due in part to endogenous TGF-beta. These results establish that resistance of melanoma cells to TGF-beta-induced growth inhibition occurs independently of intact TGF-beta receptor/SMAD-mediated transcriptional regulation. They also suggest that melanoma-derived TGF-beta may exert autocrine effects on SMAD-sensitive target genes.

Development of invasive and growth factor-independent cell variants from primary human melanomas.

Growth autonomy and high levels of invasiveness are characteristics of human melanoma cells that are metastatic in vivo. By consecutive passage through a reconstructed basement membrane, we have selected from 5 of 6 primary melanoma cell lines variants which show an up to 10-fold increase in invasiveness. The invasive variants grew more rapidly than the parental, noninvasive cells in serum- and growth factor-free medium and one of the 3 variant cell lines with the highest invasive capacity in vitro metastasized to the lungs when injected s.c. into nude mice. In a second approach, variants of 6 primary melanoma cell lines were clonally selected in medium without exogenous growth factors (protein-free medium). These selected cells showed higher invasive properties in vitro and in vivo than the parental cells. Clones of invasive and growth factor-independent cell variants were heterogenous and changed over time in the absence of selected pressure to a phenotype similar to that of parental nonselected cells. These results indicate that primary melanoma cells contain subpopulations of cells that have the phenotype of an advanced (metastatic) stage of tumor progression, but this phenotype is not stable without selective pressure.

Presence of tumor-associated antigens in epidermal growth factor receptors from different human carcinomas.

The epidermal growth factor (EGF) receptor in two colon carcinoma lines and in one vulval carcinoma line tested contains carbohydrate determinants that are recognized by monoclonal antibodies to tumor-associated antigens. These antibodies are directed to sialylated Lea and to difucosylated structures of the Y type. Cell lines which react with these antibodies express these antigens on their surface glycolipids and glycoproteins, including the EGF receptor. These unusual carbohydrates are absent in EGF receptors from normal untransformed cells, and from tumor cells which do not express these specific antigens. Although EGF receptor represents only 0.1-2% of total plasma membrane proteins of antigen-positive carcinomas, it accounts for 20-80% of total protein-associated sialylated Lea/Y type of nonsecreted carbohydrates present in these cells. The results of cell-binding, immunoprecipitation, and Western blot analyses of the antigen-positive carcinomas indicate that sialylated Lea/Y type of antigenicity is intrinsic to the EGF receptors of these cells, and that the antigen is present in receptors from both over-expressing and normal-expressing carcinomas.

Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor.

Human melanoma cells express two prominent angiogenic factors, e.g., vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF/fibroblast growth factor-2). In this study, we report on the relative contribution of these two factors to in vitro and in vivo growth of a tumorigenic melanoma cell line (WM164) and nontumorigenic, immortalized melanocytes (FM516SV). Overexpression of either cytokine significantly boosted tumorigenicity of WM164 cells in immunodeficient SCID mice. Attempting to overexpress bFGF antisense sequences produced no viable clones confirming earlier reports that autocrine bFGF is obligatory to melanoma cell survival and growth. By contrast, down-regulation of endogenous VEGF production did not affect growth of WM164 cells in vitro. In vivo expansion of WM164 cells expressing VEGF antisense was delayed but not abrogated. Forced expression of either bFGF or VEGF in immortalized but nontumorigenic melanocytes did not induce sustained tumor growth in vivo highlighting that neither of the two factors is sufficient for induction of tumorigenicity in this model system. Overexpression of either cytokine in WM164 cells led to the development of atypical large vessels but not to an increase in microvessel density. Taken together our results confirm an essential autocrine role of bFGF in human melanoma and indicate a beneficial but nonessential role of VEGF in the tumorigenic phenotype of human melanoma cells.

Identification of differentially expressed messenger RNAs in human melanocytes and melanoma cells.

The phenotype of malignant lesions is a reflection of genetic events altering the RNA and protein expression patterns of normal cells. We have investigated RNA expression patterns distinguishing normal melanocytes (FM 902), a primary melanoma cell line (WM 793), and its variant cell line (1205-LU), selected for metastatic phenotype in athymic mice. Using mRNA differential display, we identified 42 different cDNA PCR products with cell line-specific expression patterns. Direct sequence analysis matched approximately 50% of the cDNA PCR products with gene sequences accessible in DNA databases. Among the known genes, two functionally distinct groups were recognized: (a) genes encoding ribosomal and mitochondrial proteins that were predominantly up-regulated in the malignant cells; and (b) genes encoding modulators of the immune response. Among the immunomodulators, the T-cell antigen MART-1 and the protease inhibitor alpha2-macroglobulin were detected in the melanocyte cell line but not in the tumor cells. By contrast, mRNAs for the complement inhibitor CD59 and the cytokine IL-1beta were found to be overexpressed in the malignant melanoma cells. RNA slot blot hybridization on a larger panel of melanocyte and melanoma cell lines confirmed differential expression of 15 of 42 genes including MART-1, alpha2-macroglobulin, and CD59. This molecular screening approach identified also three partially characterized and three novel sequences with differential expression patterns in normal and malignant melanocytes.

Transforming growth factor beta production and responsiveness in normal human melanocytes and melanoma cells.

Previous studies have shown that some human melanoma cells express transforming growth factor beta (TGF-beta) mRNA and are growth inhibited by exogenous TGF-beta, suggesting a possible negative autocrine role for this melanoma-derived growth factor. To better understand the role of endogenous TGF-beta in the development of melanoma, we investigated patterns of TGF-beta protein production and responsiveness of human melanoma cells as compared to normal melanocytes. Both cultured melanoma cells and normal melanocytes secreted biologically inactive, latent TGF-beta protein which, upon acid treatment, became biologically active. In melanoma cells, TGF-beta production occurred constitutively, i.e., in the absence of exogenous polypeptide growth factors. By contrast, in melanocytes, TGF-beta production depended on stimulation by exogenous growth factors such as insulin-like growth factor I. Exogenous, bioactive TGF-beta 1 at picomolar concentrations inhibited tritiated thymidine uptake of normal melanocytes, whereas melanoma cells demonstrated various degrees of resistance to TGF-beta-induced inhibition of DNA synthesis. Five of six cell lines were less sensitive than any of the melanocyte lines tested, and one cell line was completely resistant to inhibitory effects of TGF-beta on DNA synthesis. In vivo selection of melanoma cells for metastatic ability in athymic mice produced a variant cell line that was resistant to TGF-beta 1-induced inhibition of DNA synthesis and proliferation. Development of TGF-beta resistance in the variant cell line was not associated with changes in TGF-beta cell surface binding. Stable transfection of melanocytes with a plasmid expressing the Simian Virus 40 large T-antigen rendered these cells resistant to growth inhibition by TGF-beta, suggesting that TGF-beta inhibits melanoma/melanocyte growth via interaction with Simian Virus 40 large T-antigen-responsive transcription elements.

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor.

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas.

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma cells.

Tumor growth modulation by a monoclonal antibody to the epidermal growth factor receptor: immunologically mediated and effector cell-independent effects.

A monoclonal antibody of IgG2a isotype (425) is described that reacts with the epidermal growth factor receptor on human cells of different tissue origins. Monoclonal antibody 425 mediates tumor cytotoxicity in vitro using mouse and human effector cells and suppresses in vivo tumor cell growth of epidermoid (A 431) and colorectal (SW 948) carcinoma-derived cell lines. The tumoricidal effects in vitro are proportional to the antigen density on target cells. At concentrations higher than 1 nM, monoclonal antibody 425 inhibits growth of epidermal growth factor receptor-bearing A 431 cells, showing an epidermal growth factor-like agonist activity on the growth properties of these cells. A 431 cultures grown in the presence of growth-inhibiting doses of antibody or epidermal growth factor reveal a clear decrease of the relative number of cells in S phase. Additionally, cells treated with the antibody show a decrease of G2-M-phase cells in some, but not all, cultures tested.

Immunotherapy of human glioma xenografts with unlabeled, 131I-, or 125I-labeled monoclonal antibody 425 to epidermal growth factor receptor.

Monoclonal antibody (mAb) 425 (IgG2a) binds to the external domain of the epidermal growth factor receptor. This determinant is highly expressed by human glioma tissues but rarely by normal brain tissues, and is absent on peripheral blood lymphocytes and bone marrow cells. The mAb exerts variable cytotoxic effects against cultured human glioma cells in conjunction with human and murine effector cells. Inhibition of growth of s.c. glioma xenografts in nude mice by the mAb may be mediated by murine macrophages or may be related to the capacity of the mAb to antagonize growth stimulation of glioma cells by epidermal growth factor. In approaches to radioimmunotherapy of human glioma with mAb 425, the 125I-labeled mAb 425 exhibited more significant antitumor effects than the 131I-labeled mAb both in vitro and in vivo in xenotransplanted nude mice. These differences may be due to enhanced nuclear damage caused by 125I-labeled versus 131I-labeled fragments following their internalization into the glioma cells. Our studies provide the rationale for immunotherapy of glioma patients with either unlabeled or 125I-labeled anti-epidermal growth factor receptor mAb 425.

Expression of melanoma-associated antigens in rapidly dividing human melanocytes in culture.

Conditions were established to induce rapid clonal growth of melanocytes from newborn foreskin. Surface antigen expression was analyzed using monoclonal antibodies derived by immunization of mice with melanoma cell, melanocyte, and placental membrane preparations. Unlike resting melanocytes in normal skin, cultured melanocytes expressed most major melanoma-associated antigens tested, e.g., nerve growth factor receptor, proteoglycan, transferrin-related Mr 97,000 protein antigen, Mr 120,000 protein, and gangliosides 9-O-acetyl GD3 and GD3. HLA-DR antigen and ganglioside GD2 were expressed at very low levels or not expressed. After several subpassages, most melanocyte cultures, including clones and melanocytes, initially sorted by rosetting with monoclonal antibody to nerve growth factor receptor, lost their characteristic bipolar morphology and expression of nerve growth factor receptor and Mr 97,000 antigen but continued to express high molecular weight proteins such as proteoglycan, Mr 130,000/105,000 and 120,000 antigen. The few melanocyte cultures that did maintain their characteristic bipolar to spindle morphology continued to express all melanoma-associated antigens and even began to express HLA-DR antigens. Melanocytes cultured in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also maintained their bipolar morphology, were often pigmented, and continued to express melanoma-associated antigens for several passages; they did not express HLA-DR antigen. Our studies indicate that rapidly proliferating melanocytes in culture undergo antigenic changes associated with malignancy.

Aberrant activation of c-kit protects colon carcinoma cells against apoptosis and enhances their invasive potential.

Multiple genetic aberrations contribute to the development of biologically aggressive, clinically malignant colorectal carcinomas (CRCs). Some of these have been linked to inappropriate signaling through the tyrosine kinase moieties of growth factor receptors. We have described previously (G. Bellone et al., J. Cell. Physiol., 172: 1-11, 1997) that human CRCs overexpress both the receptor tyrosine kinase c-kit and its ligand, stem cell factor (SCF), relative to normal mucosa cells, thus establishing an autocrine c-kit-mediated loop. In addition, we noted that exogenous SCF contributes to anchorage-independent growth of HT-29 colon carcinoma cells in semisolid medium. Here, we investigated possible roles of the c-kit/SCF autocrine/paracrine system in survival and invasive capacity of DLD-1 colon carcinoma cells. We report that SCF was required for migration and invasion of DLD-1 cells through reconstituted basement membranes (Matrigel) and up-regulated gelatinase (matrix metalloproteinase-9) activity in DLD-1 cells. Furthermore, we describe that SCF supported survival of DLD-1 cells in growth factor-deprived conditions. These results suggest multiple roles of c-kit activation in support of the malignant phenotype of DLD-1 cells related to growth, survival, migration, and invasive potential.

Inhibition of the fibroblast growth factor receptor 1 (FGFR-1) gene in human melanocytes and malignant melanomas leads to inhibition of proliferation and signs indicative of differentiation.

Fibroblast growth factor receptor (FGFR-1) is expressed as a single 3.5-kb mRNA transcript in normal human melanocytes and in malignant melanomas as determined upon Northern hybridization to a cDNA clone encoding the membrane-spanning form of the human FGFR-1. Polyclonal antisera directed against the chicken FGFR recognized a 145-kDa protein in primary and metastatic melanomas. Antisense oligodeoxynucleotides targeted against the translation start site and a splice donor-acceptor site of human FGFR-1, in addition to inhibiting the proliferation of normal human melanocytes and malignant melanomas, caused extensive dendrite formation and severe disruption of cell-cell contact--morphological changes that were not observed upon inhibition of the genes encoding basic fibroblast growth factor (bFGF) and retinoic acid-alpha receptor. Thus, unlike in the case of the ligand bFGF, expression of the FGFR-1 may represent a requisite to prevent human melanocytes and malignant melanomas from undergoing (terminal) differentiation.

Growth-regulatory factors for normal, premalignant, and malignant human cells in vitro.

Normal human cells, cells from nonmalignant proliferative lesions, and primary and metastatic tumor cells can be maintained in vitro and analyzed for requirements for growth in chemically defined media. The human melanocytic cell system with normal melanocytes, precursor nevus cells, and primary and metastatic melanoma cells has been extensively studied for the phenotypic properties of the cells, including their requirements for exogenous growth factors and other mitogens. In high calcium-containing W489 medium, normal melanocytes require four supplements: IGF-I (or insulin); bFGF, TPA, and alpha-MSH. Nevus cells are largely independent of bFGF. Depletion of TPA from medium is not as detrimental to nevus cells as it is to melanocytes, but the phorbol ester is still essential for maintenance of the typical nevic phenotype. Primary melanoma cells require at least one growth factor, IGF-I (or insulin), for continuous proliferation. On the other hand, metastatic cells of melanoma as well as of carcinomas of colon and rectum, bladder, ovary, and cervix are able to proliferate after a short adaptation period in medium depleted of any growth factors and other proteins. Doubling times of metastatic tumor cells in protein-free medium are only 30-60% longer than in FCS-containing medium. The growth autonomy of human tumor cells is apparently due to the endogenous production of growth factors. Likely candidates for autocrine growth stimulation of human tumor cells are TGF-alpha, TGF-beta, and PDGF. Melanoma and colorectal carcinoma cells express functional EGF/TGF-alpha receptors, and produce TGF-alpha, indicating that this growth factor is produced for autocrine stimulation. In addition to the use of anti-growth factor antibodies, other strategies for the inhibition of autocrine growth stimulation include mAbs to growth factor receptors, soluble receptors, receptor-mimicking antiidiotype antibodies, and active immunization against growth factors. Whether any of these therapeutic approaches is clinically feasible will need to be determined in extensive preclinical investigations.

Expression and transcriptional regulation of caspase-14 in simple and complex epithelia.

Caspase-14 is a recent addition to the caspase family of aspartate proteases involved in apoptotic processes. Human caspase-14 appears to be only weakly processed during apoptosis, and it does not cleave classical caspase substrates. Post partum, caspase-14 is prominently expressed by human keratinocytes and reportedly participates in terminal differentiation of complex epithelia. Here we provide evidence challenging the view that caspase-14 expression or processing is linked exclusively to terminal keratinocyte differentiation. We demonstrate that caspase-14 expression extended to multiple cell lines derived from simple epithelia of the breast, prostate, and stomach. In keratinocytes and breast epithelial cells, caspase-14 expression was upregulated in high-density cultures and during forced suspension culture. These effects were primarily due to transcriptional activation as indicated by reporter gene assays using a 2 kb caspase-14 promoter fragment. Importantly, caspase-14 was not cleaved during forced suspension culture of either cell type although this treatment induced caspase-dependent apoptosis (anoikis). Forced expression of caspase-14 in immortalized human keratinocytes had no effect on cell death in forced suspension nor was the transfected caspase-14 processed in this setting. In contrast to postconfluent and forced suspension culture, terminal differentiation of keratinocytes induced in vitro by Ca2+ treatment was not associated with increased caspase-14 expression or promoter activity. Our results indicate that (1) caspase-14 is expressed not only in complex but also simple epithelia; (2) cells derived from complex and simple epithelia upregulate caspase-14 expression in conditions of high cell density or lack of matrix interaction and; (3) in both cell types this phenomenon is due to transcriptional regulation.