Treatment of melanoma cells with the synthetic retinoid CD437 induces apoptosis via activation of AP-1 in vitro, and causes growth inhibition in xenografts in vivo.

Human malignant melanoma is notoriously resistant to pharmacological modulation. We describe here for the first time that the synthetic retinoid CD437 has a strong dose-dependent antiproliferative effect on human melanoma cells (IC50: 5 x 10(-6) M) via the induction of programmed cell death, as judged by analysis of cell morphology, electron microscopical features, and DNA fragmentation. Programmed cell death was preceded by a strong activation of the AP-1 complex in CD437-treated cells as demonstrated by gel retardation and chloramphenicol transferase (CAT) assays. Northern blot analysis showed a time-dependent increase in the expression of c-fos and c-jun encoding components of AP-1, whereas bcl-2 and p53 mRNA levels remained constant. CD437 also exhibited a strong growth inhibitory effect on MeWo melanoma cells in a xenograft model. In tissue sections of CD437-treated MeWo tumors from these animals, apoptotic melanoma cells and c-fos overexpressing cells were colocalized by TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) staining and in situ hybridization. Taken together, this report identifies CD437 as a retinoid that activates and upregulates the transcription factor AP-1, leading eventually to programmed cell death of exposed human melanoma cells in vitro and in vivo. Further studies are needed to evaluate whether synthetic retinoids such as CD437 represent a new class of retinoids, which may open up new ways to a more effective therapy of malignant melanoma.

Glutathione and related enzymes in tumor progression and metastases of human melanoma.

We have shown previously that overexpression of p-170 glycoprotein-mediated multidrug resistance plays only a minor role in conferring chemoresistance to human melanoma cells. In addition to membrane transporters like p-170, metabolizing enzyme systems have been implicated in altered drug sensitivity. Recently, glutathione and associated enzymes have been associated with resistance to alkylating substances, particularly in gastrointestinal and gynecologic cancers. In this study, we investigated whether increased levels of glutathione and related enzymes may play a role in chemoresistance in melanoma. Levels of glutathione, glutathione S-transferase (GST), glutathione reductase, and gamma-glutamyl transpeptidase were analyzed in melanoma and non-melanoma cell lines. In addition, 18 melanoma metastases derived from skin and lymph nodes were examined. Levels of gamma-glutamyl transpeptidase were statistically different in cells derived from melanocytic tumors compared with non-melanoma cell lines and normal cells. In addition, GST levels in metastases derived from skin or lymph nodes were significantly lower than those in permanent cell lines. However, levels of glutathione and related enzymes in metastases and cell lines fluctuated over a wide range, up to 40-fold, regardless of treatment status or origin of metastases. In a second part of the study, the expression of GST isoenzymes alpha, mu, and pi was studied by immunohistology in 10 benign nevi, 29 primary melanomas, and 39 melanoma metastases before and during chemotherapy. Expression of GST isoenzymes was increased with tumor progression, and GST pi was the strongest isoform expressed. However, no correlation was found between GST levels by immunohistochemistry and the course of tumor progression, between GST levels in metastases obtained before or during chemotherapy, or between GST levels and clinical response. These data suggest that alterations in glutathione metabolism and the expression of GST do not play a major role in resistance to chemotherapeutic drugs in melanoma.

Retinoic Acid receptor-gamma-selective retinoids exert antiproliferative effects on human-melanoma cell-growth in-vitro.

Malignant melanoma is a cancer with dramatically increasing incidence and mortality. Alternative approaches for therapy of disseminated malignant melanoma are urgently needed. In order to develop new alternatives for therapy of metastatic melanoma, we tested the biological activity of eight synthetic retinoids with high affinity and/or selectivity for the retinoic acid receptors (RAR) alpha, beta and gamma in comparison to all-trans retinoic acid, arotinoid acid and CD 367 in four human melanoma cell lines. The melanoma cell growth was not affected by most retinoids tested, however, two of the synthetic substances with high RAR-gamma selectivity (CD 437 and CD 2325) showed a dose-dependent antiproliferative effect on all melanoma cell lines tested with IC50 values between 10(-6) and 10(-7) M. A structurally closely related compound, CD 2398, lacking the carboxyl-terminal group and therefore exhibited no RAR-binding did not change melanoma cell growth behaviour. We demonstrate the expression of RAR-alpha, RAR-beta and RAR-gamma, in four human melanoma cell lines by polymerase chain reaction. Taken together, these results suggest that binding and stimulation of the RAR-gamma receptor might be a major factor in growth inhibition of human melanoma cell by retinoids in vitro. Although several of the compounds altered the cell surface expression of defined melanocytic differentiation markers, the correlation to changes in melanin content was poor. Furthermore, five out of eight retinoids modulated HLA-DR expression on human melanoma cells. The potent growth inhibitory effect of the RAR-gamma selective retinoids as well as their immunomodulating capacities opens an interesting alternative for new antiproliferative and immunomodulatory strategies in the therapy of metastatic melanoma.

Allergen inhalation challenge induces decrease of serum neutral endopeptidase (NEP) in asthmatics.

There is accumulating evidence that tachykinins are implicated in inflammation, including asthma. Therefore, we hypothesized that the neutral endopeptidase (NEP), under challenge conditions, could be affected. Serum from 21 asthmatics and six healthy volunteers was sampled before, 30, and 120 min after allergen challenge. NEP-IR was determined using an ELISA and was found in all subjects. Compared to prechallenge, no difference was seen between asthmatics and controls; however, under challenge conditions, NEP-IR in asthmatics was significantly lower (30 min, P = 0.058; 120 min, P = 0.0017, respectively). This finding supports indirectly the hypothesis that tachykinins are released during allergen exposure, and suggests a regulatory role of NEP.

Effects of various synthetic retinoids on proliferation and immunophenotype of human melanoma cells in vitro.

Since response rates in human melanoma are low with currently available therapeutic modalities, we have reevaluated the potential usefulness of retinoids as new alternatives for therapy of metastatic melanoma. Nine synthetic retinoids with high affinity and/or selectivity for the retinoic acid receptors (RAR) alpha, beta, and gamma were studied in comparison to all-trans retinoic acid (RA) for their in vitro effects on melanoma cell proliferation and for their immunomodulating capacities using four human melanoma cell lines. Eight out of ten retinoids tested had no effect on melanoma cell growth, whereas the remaining two compounds with high RAR-gamma selectivity (CD437 and CD2325) showed a dose-dependent antiproliferative effect on all melanoma cell lines with IC50 (concentration inhibiting response by 50%) values between 10(-6) and 10(-7)M. Further analyses showed that paracrine-mediated tumor cell growth inhibition such as induction of transforming growth factor (TGF)-beta described as one mechanism of retinoid action and enzyme systems such as tyrosinase and monoamine oxidase were not involved in mediating the antiproliferative effects exerted by the two retinoids. Four of nine retinoids modulated HLA-DR expression on human melanoma cells, and expression levels of intercellular adhesion molecule 1 (ICAM-1) was increased by another subset of compounds. These effects were, however, not correlated to the receptor selectivity of the retinoids. The potent growth inhibitory effect of the RAR-gamma-selective retinoids and the immunomodulating capacities of the retinoids open an interesting alternative for new antiproliferative and immunomodulatory strategies in the treatment of metastatic melanoma.

In vitro sensitivity of human melanoma cells to chemotherapeutic agents and interferons.

The purpose of our study was to evaluate systematically the anti-proliferative effects of eight chemotherapeutic drugs as well as of four recombinant interferons (IFNs) (alpha-2a, alpha-2b, beta, gamma). All drugs and IFNs were tested separately and in combination at several concentrations on four human melanoma cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium (MTT) test. In all cases, drug inhibitory concentrations of chemotherapeutic agents required to kill 25% of melanoma cells (IC25) in vitro were in the range of the maximal achievable plasma peak level in vivo. Sensitivity to the anti-proliferative action of bleomycin, DTIC, doxorubicin, cisplatin and carboplatin was similar for all melanoma cell lines, whereas cell lines exposed to 5-fluorouracil (5-FU), vindesine and fotemustine differed up to 26-fold in their sensitivity. Studies with IFN showed that IFN-beta and IFN-gamma proved to be more antiproliferative than IFN-alpha in a dose-dependent fashion in all cell lines. However, the ability of IFNs to improve cytotoxicity of chemotherapeutic agents was limited. Pre-incubation of melanoma cells with IFN as well as exposure to IFN after incubation with the drugs showed mainly additive effects (231/256). These results confirm the high chemoresistance of human melanoma cells, independently of the drug chosen. Combinations of chemotherapeutic agents with IFN will provide additional therapeutic benefit, but are unlikely to change the overall high chemoresistance of human melanoma cells.

Human melanoma cell lines selected in vitro displaying various levels of drug resistance against cisplatin, fotemustine, vindesine or etoposide: modulation of proto-oncogene expression.

Melanoma cells often display a multidrug-resistant phenotype, but the mechanisms involved are largely unknown. In order to establish a reproducable model system for studying the exact mechanisms conferring chemoresistance, we selected drug-resistant sublines in vitro derived from one parental human melanoma (MeWo) cell line. Four commonly used chemotherapeutic drugs (vindesine, etoposide, fotemustine, cisplatin) with different modes of action were choosen and stable sublines exhibiting four different levels of resistance against each drug were selected by continuous exposure over two years. Analysis of the drug-resistant sublines regarding their pharmacological characteristics and cross-resistance pattern revealed an up to 26-fold increased relative resistance against the alkylating agent fotemustine (MeWoFOTE) and an up to 35.7-fold increased relative resistance against topoisomerase-II-inhibiting etoposide (MeWoETO). Cisplatin selection (MeWoCIS) resulted in a 6-fold higher resistance compared to parental MeWo cells, whereas vindesine exposure (MeWoVIND) increased relative resistance up to 10.2-fold. Sublines selected separately for resistance to the DNA-damaging agents fotemustine, cisplatin and etoposide demonstrated strong cross-resistance. In comparison to the parental cell line drug-resistant sublines showed altered expression patterns of proto-oncogenes. Levels of p53 mRNA decreased with increasing resistance to vindesine, etoposide and fotemustine. Expression of bcl-2 family members (bax, bcl-x) was modulated by fotemustine, etoposide and cisplatin. In addition the expression of members of the fos (c-fos) and jun (c-jun, jun-D) gene family encoding transcription factors of the AP-1 complex was altered in all drug-resistant sublines. The pattern of expression varied with the inducing stimulus and this was paralleled by changes in the transactivation potential of AP-1. Our results reinforce the central role of AP-l in drug resistance probably through its participation in a programmed cellular stress response.

Antioxidative enzymes in human nasal mucosa after exposure to ozone. Possible role of GSTM1 deficiency.

OBJECTIVE AND DESIGN: Epithelial antioxidative enzymes (AOEs) are thought to be a first line of defense against reactive oxygen species as they are upregulated after exposure to ozone according to animal studies. We analysed the activities of the AOEs catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), superoxide dismutase (SOD) and glutathione-S-transferase (GST) in a tissue culture of human nasal mucosa and analysed the influence of GSTM1 polymorphism on AOE regulation. METHODS: Tissue biopsies of 20 subjects were incubated for 24 h with and without 120 ppb ozone. Activities were assayed to determine what enzymatic changes had taken place, both overall and in regard to GSTM1 status. RESULTS: Activities for GPX (p = 0.272) and SOD (p = 0.291) were found increased after ozone exposure. GSTM1-deficient patients showed a significantly enhanced upregulation of SOD activity (p = 0.011) compared to GSTM1 carriers. CONCLUSION: Our findings suggest that GSTM1-deficiency has an impact on AOE-regulation after ozone exposure.

Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation.

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.

Upregulation of TNF-alpha and IL-3 expression in lesional and uninvolved skin in different types of urticaria.

BACKGROUND: Although mast cells are known to secrete a broad spectrum of proinflammatory and immunomodulatory cytokines, the role of these molecules in mast cell-dependent cutaneous inflammation is not clear. OBJECTIVE: We decided to study biopsy specimens from lesional and nonlesional skin of patients with acute, chronic recurrent, delayed pressure, and cold urticaria; from fleeting wheals of prick test reactions to allergens; and from normal skin of nonallergic subjects. METHODS: Cryostat sections were stained by immunohistochemistry with antibodies against IL-3, IL-8, TNF-alpha, and mast cell-specific tryptase. In serial sections with tryptase and each cytokine, reactivity of mast cells was studied as well. RESULTS: Compared with normal skin and prick test reactions, immunoreactivity for TNF-alpha and IL-3 was significantly increased on endothelial and perivascular cells of the upper dermis in all urticaria lesions. In nonlesional skin comparable upregulation was noted on endothelial cells and for TNF-alpha on perivascular cells of patients with delayed pressure urticaria. In addition, TNF-alpha was expressed throughout the epidermis in lesional and nonlesional skin of patients with all types of urticaria, but not in normal control subjects. Sequential biopsy specimens from patients with cold urticaria showed upregulation of TNF-alpha and IL-3 on endothelial cells 30 minutes after elicitation of lesions with an ice cube. In contrast to these findings, epidermal immunoreactivity, as well as endothelial and perivascular cell expression of IL-8, were only slightly altered in urticaria compared with normal skin. In sequentially stained sections, few tryptase-positive mast cells reacted to TNF-alpha, few reacted to IL-3 in pressure urticaria only, and practically none stained for IL-8. CONCLUSION: These findings suggest that the cytokines studied here are involved in the pathology of urticaria, possibly by inducing subthreshold inflammation in endothelial cells of uninvolved skin.

Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study.

Cytokine gene transfer into tumor cells has been shown to mediate tumor regression and antimetastatic effects in several animal models via immunomodulation. Therefore, clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumor cells. We inserted the genes coding for the p35 and p40 chain of interleukin-12 (IL-12) in two independent eukaryotic expression vectors and transduced melanoma cells of 15 different primary tumor cultures with both plasmids by a ballistic gene transfer approach. Secreted IL-12 demonstrated strong bioactivity by inducing interferon-gamma release from peripheral blood lymphocytes upon coculture with cell culture supernatants after IL-12 gene transfer which could at least partly be blocked by IL-12-specific antisera. Further enrichment of transduced tumor cells by magnetic separation directly after gene transfer increased cytokine secretion from a mean of 119 pg in the unsorted to 507 pg IL-12 (24 h/10(8) cells) in the magnetically enriched cell fraction. Irradiation of these cells led to a further elevation of secreted IL-12 (mean 987 pg). Elevated IL-12 levels were detected over 7 days after irradiation in vitro. In a subsequent first clinical phase I study six patients with metastatic melanoma were vaccinated with autologous, interleukin-12 gene-modified tumor cells. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by ballistic gene transfer, irradiated and were then injected subcutaneously (s.c.) at weekly intervals. Clinically, there was no major toxicity except for mild fever. All patients completed more than four s.c. vaccinations over 6 weeks and were eligible for immunological evaluation. Post-vaccination, peripheral mononuclear cells were found to contain an increased number of tumor-reactive proliferative as well as cytolytic cells as determined by a limiting dilution analysis in two patients. Two patients developed DTH reactivity against autologous melanoma cells and one had a minor clinical response. Biopsies taken from that patient's metastases revealed a heavy infiltration of CD4+ and CD8+ T lymphocytes. In conclusion, vaccination induced immunological changes even in a group of advanced, terminally ill patients. These changes can be interpreted as an increased antitumor immune response.

Vaccination with IL-7 gene-modified autologous melanoma cells can enhance the anti-melanoma lytic activity in peripheral blood of patients with a good clinical performance status: a clinical phase I study.

Recently, cytokine gene transfer into tumour cells has been shown to mediate tumour regression in animal models via immunomodulation. Consequently, a number of clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumour cells. Here, we report the results of a clinical phase I trial using for the first time autologous, interleukin 7 gene-modified tumour cells for vaccination of ten patients with disseminated malignant melanoma. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by a ballistic gene transfer technique and were then injected after in vitro irradiation s.c. at weekly intervals. Clinically, there was no major toxicity except for mild fever, and no major clinical response towards vaccination was observed. Eight of ten patients completed the initial three s.c. vaccinations and were eligible for immunological evaluation. Post vaccination, peripheral mononuclear cells (PBMCs) were found to contain an increased number of tumour-reactive proliferative as well as cytolytic cells, as determined by a limiting dilution analysis. In three of six patients, the frequencies of anti-melanoma cytolytic precursor cells increased between 2.6- and 28-fold. Two of these patients showed a minor clinical response. Analysis of the autologous tumour cell vaccines regarding IL-7 secretion after gene transfer, HLA class I and class II cell surface expression, secretion of immunosuppressive mediators (TGF-beta1, IL-10) and various melanoma-associated tumour antigens revealed a very diverse expression profile. In conclusion, vaccination using gene-modified autologous melanoma cells induced immunological changes in a group of advanced, terminally ill patients. These changes can be interpreted as an increased anti-tumour immune response. However, immunological modulation was most pronounced in patients in good physical condition. Therefore, patients with minimal tumour load or minimal residual disease might preferentially benefit from tumour cell vaccination in further studies. In order to evaluate the effects of the cytokine gene-modified tumour cell vaccines more precisely, an antigenically better defined vaccine is needed.