CTGF is a therapeutic target for metastatic melanoma.

Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.

Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells.

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40.

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

APC-dependent changes in expression of genes influencing polyamine metabolism, and consequences for gastrointestinal carcinogenesis, in the Min mouse.

The colorectal mucosa of pre-symptomatic individuals with familial adenomatous polyposis (FAP) contains elevated levels of the proliferation-associated polyamines. The Min mouse, like humans with FAP, expresses an abnormal genotype for the APC tumor suppressor gene. In order to determine how APC mutation influences intestinal tissue polyamine content, we measured steady-state RNA levels of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, antizyme (AZ), a protein which negatively regulates ODC, and the spermidine/spermine N(1)-acetyltransferase (SSAT), the first enzyme in polyamine catabolism. RNA content was increased 6- to 8-fold in both the small intestine and colon for ODC, decreased significantly in the small intestine but not the colon for AZ and was not statistically different in either intestinal tissue for SSAT in Min mice compared with normal littermates. Consistent with the changes in ODC and AZ gene expression, small intestinal, but not colonic, polyamine content was elevated in Min mice compared with normal littermates. Treatment of Min mice with the specific ODC inhibitor difluoromethylornithine (DFMO) suppressed small intestinal, but not colonic, polyamine content and tumor number. These data indicate that small intestinal tissue polyamine content is elevated in Min mice by a mechanism involving APC-dependent changes in ODC and AZ RNA. Further, ODC enzyme activity, which is influenced by both ODC and AZ RNA levels and inhibited by DFMO, is consequential for small intestinal tumorigenesis in this model. In the FAP population, DFMO may be of value in the chemoprevention of small intestinal adenocarcinoma that remains a risk following colectomy.

Pigmented uveal tumours in a transgenic mouse model.

AIMS/BACKGROUND: The authors have developed transgenic mouse strains at the Arizona Cancer Center using a tyrosinase promoter to target expression of the mutated T24 Ha-ras gene in melanin producing cells. Histopathology and electron microscopy (EM) were performed to characterise the intraocular tumours observed phenotypically. METHODS: Transgenic TPras mice (n = 8) and normal, age matched control mice (n = 6) were sacrificed at 3 weeks, 6 weeks, 7 weeks, 4 months, 5 months, 9 months, and 11 months. Six were processed in formalin for light microscopic examination and eight in a glutaraldehyde/formalin solution for electron microscopic examination. RESULTS: Six of the TPras mice were found to have bilateral pigmented melanocytic/RPE proliferations of the uveal tract. The cytological characteristics of the tumours included low nuclear to cytoplasmic ratios (N:C ratios), bland nuclei, and abundant intracytoplasmic melanin. By EM two populations of cells were identified, including spindle-shaped cells with round to oval melanin granules and cuboidal cells with apically located, cigar-shaped, melanin granules, and basement membrane formation. A 3 week and an 11 month old TPras mouse had a higher grade, bilateral, melanocytic proliferation of the uveal tract which, although not metastatic, was morphologically melanoma. Cytological features included increased N:C ratios, nuclear pleomorphism, and prominent nucleoli. The uveal tract was normal in both eyes in all of the control animals. CONCLUSION: Pigmented intraocular tumours developed in transgenic strains of mice that express a mutated Ha-ras gene in melanin producing cells. The morphology was most consistent with a melanoma in two of the mice and a benign melanocytic/RPE proliferation in the remaining mice.

UVB irradiation-induced activator protein-1 activation correlates with increased c-fos gene expression in a human keratinocyte cell line.

The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation. Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.

Different bile acids exhibit distinct biological effects: the tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation.

Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.

Chromosomal and genetic alterations of 7,12-dimethylbenz[a]anthracene-induced melanoma from TP-ras transgenic mice.

The TP-ras transgenic mouse line expresses an activated human T24 Ha-ras gene with a mutation in codon 12, regulated by a mouse tyrosinase promoter. The transgene is expressed in melanocytes of the skin, eyes, and brain. The mice develop cutaneous melanoma when treated with 7,12-dimethylbenz[a]anthracene. Cell lines have been generated from the cutaneous tumors and metastatic lesions. By using fluorescence in situ hybridization with mouse whole chromosome paints, the cell lines were characterized for chromosomal abnormalities. Key findings in the tumor cells included translocations of chromosome 4 and alterations in chromosome 6. One tumor cell line contained a double translocation involving chromosomes 3 and 6. To extend the results of the chromosome 4 painting, Southern analysis of the p15INK4B, p16INK4A, and p19INK4D genes was performed. Our data indicated that there were homozygous and partial allelic deletions and polymorphisms in the region of chromosome 4 containing these genes, resulting in the absence or reduced expression of the p16 product. These findings are similar to those reported for human melanoma, and the TP-ras transgenic mouse may therefore be a valuable model for studying novel strategies for melanoma prevention and treatment.

Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59.

We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.

Melanocyte mediated paracrine induction of extracellular matrix degrading proteases in squamous cell carcinoma cells.

The expression of extracellular-matrix (ECM)-degrading proteases has been shown to be necessary for invasion of tumor cells into surrounding tissue. For several tumor types, overexpression of these proteases is dependent upon interactions with adjacent fibroblast cell populations. We previously demonstrated activation of matrix metalloprotease (MMP) and urokinase-type plasminogen activator (uPa) expression in a coculture model consisting of squamous cell carcinoma cells (SCC) with dermal fibroblasts. In the present study we have examined whether melanocytes, which are known to interact closely with keratinocytes of the basal epidermal layer, might influence ECM-degrading protease expression in SCC cells as well. Upon coculture of the human SCC cell line II-4 with the nontumorigenic mouse melanocyte cell line Melan-a or treatment of II-4 cells with Melan-a conditioned media, induction of expression of the MMP matrilysin and uPa was observed. In contrast, no induction was observed for stromelysin-1 or 92-kDa type IV collagenase. Matrilysin/uPa-inducing activity was found to act at the level of gene transcription for both matrilysin and uPa and was ubiquitously expressed among six different human melanocytic cell strains/lines, ranging from primary normal melanocytes to cell lines established from metastatic melanoma lesions. These data demonstrate that melanocytic cells can exert a paracrine influence in SCC cells on the expression of specific proteases involved in ECM turnover and tumor invasiveness.

Hyperpigmentation and melanocytic hyperplasia in transgenic mice expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter.

The tyrosinase promoter has been used to target expression of the mutated human T24 Ha-ras oncogene in pigment-producing cells of transgenic mice. Two independent founder mice carrying the transgene survived and showed the same distinct phenotype of mutated coat color, deeply pigmented skin with multiple nevi, and twirling behavior. The offspring of one of these founders were developed into a line that stably expressed the same phenotype. Histopathological analysis of the tissues revealed hyperpigmentation and/or melanocytic hyperplasia in the skin, eyes, inner ear, and meningeal membranes in the brain. Reverse transcriptase-polymerase chain reaction analysis revealed expression of the transgene in skin, brain, and spleen. We propose that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.

Paracrine factor and cell-cell contact-mediated induction of protease and c-ets gene expression in malignant keratinocyte/dermal fibroblast cocultures.

The purpose of this study was to characterize stromal-epithelial interactions that result in induction of protease gene expression in squamous cell carcinoma of the skin. Coculture of the human squamous cell carcinoma cell line II4 with primary human foreskin fibroblasts was observed to induce mRNA expression of urokinase-type plasminogen activator (uPa), matrilysin, 92-kDa type IV collagenase, and c-ets, a transcriptional activator of several genes within the serine and matrix metalloprotease families. uPA and c-ets induction were localized to the fibroblast cell population. uPa induction was found to be dependent upon cell-cell contact with the tumor cell population, whereas c-ets induction was due to a combination of cell-cell contact and a tumor cell-derived soluble factor. In contrast, matrilysin induction localized to the tumor cells and was shown by Northern and Western analyses to occur in response to a fibroblast-derived soluble factor. These data demonstrate that both paracrine factors and cell-cell contact between stromal fibroblasts and epithelial tumor cells can influence protease gene expression.

v-jun oncogene suppresses both phorbol ester-induced cell invasion and stromelysin gene expression in a mouse papilloma cell line.

The viral jun (v-jun) oncogene encodes a transcription factor that can participate in the transactivation of genes through the AP-1 complex. Evidence indicates that the ability of v-jun to transform cells and stimulate transcription depends on the cell type. We have asked whether expression of the v-jun gene in benign tumor forming mouse keratinocytes that already express an activated c-rasHa oncogene would cause malignant progression. Our results showed that the v-jun transfection did not result in malignant progression; instead, we made the unexpected observation that the ability of these cells to invade reconstituted basement membrane matrix (in vitro) in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was suppressed. This phenomenon could, in part, be explained by the suppression of the induction by phorbol ester of expression of the metalloproteinase, stromelysin (transin). Of interest was the finding that 12-O-tetradecanoylphorbol-13-acetate induction of other cellular genes known to be regulated by AP-1 was not inhibited in the benign tumor cells expressing v-jun.

Protein kinase C beta expression in melanoma cells and melanocytes: differential expression correlates with biological responses to 12-O-tetradecanoylphorbol 13-acetate.

Normal human melanocytes require 12-O-tetradecanoylphorbol 13-acetate (TPA) for prolonged growth in vitro. In contrast, the growth of human malignant melanoma cells is often inhibited by TPA. In this study, we have confirmed and extended these observations. Since protein kinase C (PKC) is an important mediator of the effects of TPA, we have investigated the nature of this differential growth response by examining PKC expression and activity in primary cultures of human neonatal melanocytes and metastatic melanoma cell strains. PKC, when measured by immunoreactivity or a functional assay, was found to be more abundant in melanoma cells than in melanocytes. When specific isotypes were examined by Northern analysis, PKC-alpha and -epsilon were expressed in both melanocytes and melanoma. PKC-beta was expressed in melanocytes, but was undetectable by Northern analysis in 10 out of 11 melanoma cell strains. Southern analysis revealed that no gross deletions or rearrangements of the PKC-beta gene had occurred. These data suggest that down-regulation of the PKC-beta gene occurs frequently during the process of transformation of melanocytes. Furthermore, differential expression of PKC isotypes may explain the different effects of TPA on melanocyte and melanoma cell growth.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in murine skin by systemic effects of ultraviolet irradiation.

Systemic effects of UVB irradiation (280-320 nm) have been shown to prevent subsequent chemical tumorigenesis induced by an initiation-promotion protocol. The present investigation was designed to determine whether initiation or promotion is prevented by UV irradiation. Groups of 25 B6D2F1/J mice received 12 weeks of intermittent dorsal UVB radiation treatments administered before, or 3 weeks after, initiation with a single application of 7,12-dimethylbenz[a]anthracene on the ventral skin. All mice were promoted ventrally with 5 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) applied three times weekly throughout the experiment. UV irradiation consisted of five 30-min exposures per week to a bank of 6 Westinghouse FS40 sunlamps. UV irradiation applied before or after initiation resulted in a decrease of 18-16 tumors per group of 25 mice, for a reduction of 61 and 50%, respectively, at 24 weeks after the first TPA treatment. Thus, prevention of tumor development was similar whether the UV influence was present or not during initiation. This finding suggests that the UV prevention of promotion could account for UV inhibition of skin tumors induced by an initiation-promotion regimen. Consistent with this concept, pretreatment of mice with dorsal UVB radiation was found to reduce DNA synthesis after exposure to TPA by 46%, although it did not decrease tritiated benzo[a]pyrene binding to DNA, in ventral epidermis. Thus, UVB irradiation systemically reduced TPA-induced tumor promotion in murine skin.

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity.

Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE.

Protein-antigen specific Ia-restricted cytolytic T cells: analysis of frequency, target cell susceptibility, and mechanism of cytolysis.

We previously showed that cloned, antigen-specific, Ia-restricted L3T4a+ T cell lines can be cytolytic for antigen-pulsed B cell lymphoma targets. Such cells can also, under different experimental conditions, activate B cells to proliferate and secrete immunoglobulin. In the present experiments, we show that this functional phenotype is a common one among a panel of cloned T cell lines. In keeping with this finding, freshly isolated, antigen-activated lymph node T cells show similar functional properties. Such cytolytic L3T4a+ T cells differ from classical H-2K/D-restricted cytolytic T cells in two distinct ways. First, Ia-restricted cytolytic T cells can kill bystander targets, whereas H-2K/D-specific cytolytic T cells do not. Second, in testing a panel of target cells by using lectin-mediated cytolysis, Ia-restricted cytolytic clones reveal large differences in target cell susceptibility, whereas all targets are similarly susceptible to H-2K/D-specific killer cells. Finally, evidence is presented that both direct and bystander killing effected by L3T4a+ T cells are mediated by the same soluble factors, in that there is a strong positive correlation of these two activities for individual cloned lines. The relevant mediators appear to be lymphotoxin and IFN-gamma, although the latter molecule by itself is not cytolytic on our target lines.

A comparison of lysis mediated by Lyt 2+ TNP-specific cytotoxic-T-lymphocyte (CTL) lines with that mediated by rapidly internalized lymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing.

A series of Lyt 2+, trinitrophenyl (TNP)-specific T-cell lines are shown to lyse 51Cr-labeled target cells in an antigen-specific, H-2-restricted fashion in a 4-hr assay. These cells also produce lymphotoxin, in addition to other factors, upon stimulation with TNP-haptenated syngeneic splenocytes. A technique for introducing macromolecules into the cytoplasm of fibroblasts by inducing the cells to pinocytose the molecule in hypertonic medium, and then lysing the newly formed pinocytic vesicles with a mild hypotonic shock was used to assess the role of soluble mediators in the cytotoxic T lymphocyte (CTL)-mediated lytic process. The technique itself has little effect on cell growth rate or viability. A minimum of 24 hr, and more frequently 48-72 hr is required for lymphotoxin to manifest it's lethal effect when it is merely included in the culture medium of growing fibroblasts. In contrast, supernatant fluids from the Lyt 2+ cells kill 51Cr-labeled fibroblasts in a dose-dependent fashion during a 4-hr assay when they are rapidly internalized via the osmotic procedure. The data serve as preliminary evidence of a role for soluble mediators such as lymphotoxin in T-cell-mediated lysis, and suggest that the cytotoxic-T-cell lethal hit may include a mechanism for rapidly internalizing a toxin into appropriate target cells.