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D Schadendorf

Publications and source records attributed to D Schadendorf.

At least 109 records · Page 6Linked to original sources
PubMed1995

Serum manganese superoxide dismutase is a new tumour marker for malignant melanoma.

Malignant melanoma in its disseminated stage is incurable. The most widely accepted criteria for the prognostic evaluation of melanoma are histopathological and clinical parameters, and the identification of additional simple, serological tumour markers is thus of paramount importance. Manganese-containing superoxide dismutase (MnSOD) belongs to a family of metalloproteins that catalyse the metabolization of oxygen radicals in order to protect these cells from radical damage. In patients with epithelial ovarian carcinomas, serum MnSOD levels have been shown to be elevated in accordance with the progression of their clinical disease. Recently, an overexpression of MnSOD was shown to suppress the malignant phenotype of human malignant melanoma cells. Therefore, we determined serum MnSOD concentrations in 33 patients with malignant melanoma at different clinical stages. Whereas MnSOD serum levels in normal subjects (n = 11) and in dermatological patients with type I allergies (n = 10) or chronic non-allergic urticaria (n = 7) were below 200 ng/ml, the MnSOD serum concentrations in melanoma patients were statistically elevated in all clinical stages compared with normal (p < 0.005). These data suggest that elevated MnSOD serum concentrations correspond to tumour load and correlate with progression of malignant melanoma. Measurement of MnSOD serum levels might therefore provide a sensitive tool for monitoring the clinical course of melanoma.

Adult↗
PubMed1995

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.

Glutathione↗
PubMed1995

Expression of the c-kit receptor in hypomelanosis: a comparative study between piebaldism, naevus depigmentosus and vitiligo.

In order to investigate possible alterations in c-kit protein expression on epidermal melanocytes in different hypopigmentary disorders, we have examined skin specimens from one patient with piebaldism, one patient with naevus depigmentosus, and five patients with vitiligo. Cryosections were examined by immunohistochemistry using monoclonal antibodies against the c-kit protein (YB5.B8) and melanosomes (TA99). In piebaldism, hypomelanotic epidermis contained only a few TA99-positive epidermal melanocytes and no detectable c-kit protein, whereas in naevus depigmentosus the expression of c-kit protein was strong, and TA99 immunoreactivity was faint. In vitiligo lesions, no epidermal immunoreactivity for melanosomes or c-kit protein was found. Normally pigmented skin of all patients showed immunoreactivity of epidermal melanocytes for both c-kit protein and melanosomes. Different hypomelanotic lesions can thus be differentiated by absent melanocyte c-kit protein and low or no expression of melanosomal marker in piebaldism, normal c-kit but low melanosome expression in naevus depigmentosus, and the absence of all melanocyte markers in vitiligo.

Adult↗
PubMed1995

P-glycoprotein expression in primary and metastatic malignant melanoma.

Metastatic malignant melanoma is notoriously resistant to chemotherapeutic agents, but the exact mechanisms involved in this drug resistance are unknown. One recently defined major mechanism of multidrug resistance involves the overexpression of P-glycoprotein on cell membranes. In order to evaluate the significance of this putative drug efflux pump for chemoresistance of malignant melanoma, five different antibodies were employed to examine P-glycoprotein expression on tissue from 33 primary malignant melanomas and 35 metastases, before and after chemotherapy, using immunohistological techniques. The expression of P-glycoprotein was low on primary cutaneous melanomas (three of 33), and on metastases (one of 35). Normal tissue in and around the melanoma showed reactivity of endothelial cells, stromal cells and eccrine sweat glands with several antibodies tested. Chemotherapy with drugs commonly used in metastatic melanoma, including agents known to induce P-glycoprotein expression in other tumours (vindesine, cisplatin) had no effect on P-glycoprotein expression in human melanoma metastases. The high chemoresistance of human melanoma cells in vitro and in vivo is probably not mediated via P-glycoprotein, and other possible mechanisms involved will have to be explored in future studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Differential promoter activity in benign and malignant human cells of skin origin.

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.

Carcinoma↗
PubMed1995

Interferon-gamma downregulates epidermal growth factor receptors on human melanoma cells.

Unregulated expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation and has been shown to be associated with melanocytic tumor progression. Modulation of such a receptor by pharmacological agents could therefore be of clinical interest. We have studied EGF-R expression, its response to epidermal growth factor (EGF) and modulation effects by interferon gamma (IFN gamma) on human melanoma cells. Addition of EGF, anti-EGF and anti-EGF-R antibodies had no effect on proliferation of six melanoma cell lines tested. We report in this communication that EGF-R expression on human melanoma cells can be modulated by IFN-gamma. In the melanoma cell lines treated with IFN gamma, proliferative behavior was not affected; however, we demonstrate a downregulation of EGF-R expression on the protein level, by immunohistochemistry and flow cytometric analysis, and an accumulation of EGF-R mRNA by Northern blot analysis. The results suggest that IFN gamma downregulates EGF-R expression at a posttranscriptional level on human melanoma cells. This EGF/EGF-R interaction and its modulation by IFN gamma on human melanoma cells needs to be further clarified regarding its in vivo significance for the treatment and prognosis of malignant melanoma.

Blotting, Northern↗
PubMed1995

Interleukin-6 expression in the skin of patients with lupus erythematosus.

It has been proposed that interleukin-6 may play a role in the pathogenesis of autoimmune diseases like lupus erythematosus. We have therefore investigated the immunoreactivity of IL-6 in 32 skin biopsies of 23 patients suffering from chronic discoid lupus erythematosus (n = 16), subacute cutaneous lupus erythematosus (n = 5) and systemic lupus erythematosus (n = 5) as well as in uninvolved skin (n = 6) and in normal skin from healthy volunteers (n = 3). Increased immunohistochemical staining was detectable in 14 of 26 biopsies from lesional skin. The remaining biopsies from lesional, non-lesional and normal skin displayed only minimal or no reactivity, but 8 out of 12 lupus erythematosus patients had been pretreated with local or systemic antiinflammatory drugs. Irrespective of the LE subtype, immunolabelling was generally most intense in the basal layer of the epidermis, with additional intense suprabasal staining in sections from 2 of 5 SLE patients. Preferential production of IL-6 in the lower parts of the epidermis was confirmed by RNA in situ hybridization. No correlation was found between the deposition of immunoglobulins and complement at the dermo-epidermal junction and IL-6 expression in keratinocytes. These data suggest that IL-6 may be involved in LE although its exact role in the pathogenesis of the disease needs to be further elucidated.

Adult↗
PubMed1995

Membrane transport proteins associated with drug resistance expressed in human melanoma.

Melanoma cells often display a multidrug-resistant phenotype, but the mechanisms involved are largely unknown. We have studied here the recently identified transport-associated proteins, MRP and LRP, and the well-known drug resistance marker P-glycoprotein using a panel of 16 human melanoma cell lines and 71 benign and malignant melanocytic tissue samples. By flow cytometry and immunohistochemistry, expression of P-glycoprotein was not detectable on the protein level in the 10 cell lines analyzed, although by reverse transcriptase polymerase chain reaction, MDR-1 gene expression was demonstrated in 2 of 10 cell lines. In addition, immunohistology revealed P-glycoprotein expression in only 1 of 71 melanocytic lesions. In contrast, MRP was detected in a subset of melanoma cell lines by reverse transcriptase polymerase chain reaction and immunohistology (4 of 10). LRP expression was observed in 8 of 10 melanoma cell lines by immunochemistry and in 10 of 10 by reverse transcriptase polymerase chain reaction. Furthermore, MRP was detected immunohistologically in almost 50% of primary and metastatic melanoma specimens, although no significant differences were found between metastases taken before or after chemotherapy. Expression of LRP was detected in a subset of nevi with nevus cells exhibiting up to 25% positive LRP reactivity. In 13 of 21 primary melanomas and 23 of 37 metastases, more than 25% of tumor cells were stained by the LRP-56 monoclonal antibody. Particularly in the group of metastases with more than 50% of LRP-positive cells, 7 of 11 of the metastases had been previously exposed to chemotherapeutic drugs. Although the expression of membrane transport proteins may explain only the chemoresistance toward lipophilic, natural compounds and not resistance against alkylating agents, the lack of P-glycoprotein expression after chemotherapeutic treatment and the significant expression of MRP and LRP in melanoma cells provide first insights into the drug-resistant phenotype in melanoma. Additional studies analyzing the role of MRP and LRP in chemoresistance of melanoma are warranted.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Chemosensitivity testing of human malignant melanoma. A retrospective analysis of clinical response and in vitro drug sensitivity.

BACKGROUND: Clinical response rates in the treatment of patients with disseminated malignant melanoma are low and unpredictable. Several reports have documented that clonogenic assay systems for in vitro drug testing are capable of predicting resistance to therapy in vivo and might provide guidelines to improve clinical response rates. METHODS: Specimens from metastatic lesions of patients with malignant melanoma, predominantly from lymph nodes and skin, were disaggregated, exposed to a panel of 10 cytotoxic drugs for 1 hour, and subsequently cultured in agarose. Effects were calculated by the ability to form tumor colonies compared with an untreated control after 7-14 days. A retrospective comparison between the in vitro drug testing result and clinical response was possible in 19 cases. RESULTS: An average of 7.3 drugs per specimen were tested. A high degree of resistance was observed against all cytostatic agents studied independently of the tumor site. In 47 of 181 in vitro drug tests, tumor colony formation was reduced by 30-50%; in 17 of 181, the reduction was more than 50%. A retrospective analysis showed no clinical response in 11 cases and one mixed response in which patients received drugs that had been shown to be "resistant" in vitro. CONCLUSIONS: These results support the concept that in vitro drug testing promises to help avoid treatment with ineffective drugs and their associated toxic side effects. Furthermore, it may increase the likelihood of obtaining a clinical response in the treatment of disseminated malignant melanoma. The major limitation in the treatment of malignant melanoma is the lack of availability of effective agents for treatment.

Antineoplastic Agents↗
PubMed1994

[Drug resistance of malignant melanoma. Mechanisms and possible modulation].

Response rates of metastatic malignant melanoma to cytostatic treatment are disappointingly low. Although immunomodulators such as interferons are more commonly being used in combination with cytostatics, no major breakthrough has been achieved. The mechanisms underlying the high chemoresistance of melanoma cells are so far ill-defined, and investigations are only just being initiated. Several mechanisms of chemoresistance have, however, been studied with other tumours and might be relevant for human melanoma: (1) "Classical" multidrug resistance, determined by the expression of the p-glycoprotein which resembles a membrane pump that eliminates natural and synthetic agents from the cell interior. Different drugs, including calcium antagonists, interfere with its function and can thus modulate chemoresistance. Preliminary data from investigations of these mechanisms indicate that p-glycoprotein is not, however, involved in the multidrug resistance of malignant melanoma. (2) Detoxification, involving glutathione-S-transferases (GST). GST are a multigene family of enzymes which inactivate alkylating agents by conjugation to glutathione. Their relevance for chemoresistance in melanoma has not yet been clarified. (3) Topoisomerase II, which is involved in DNA recombination and DNA transcription events and represents the target of several inhibitory cytotoxic agents. Low levels of the enzyme render cells resistant to the action of specific drugs. Again nothing is yet known regarding the relevance of this mechanism in human melanoma. Further studies of these potentially important resistance mechanisms are thus urgently needed in order to develop more effective therapies for advanced malignant melanoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Lysis of allogeneic and autologous melanoma cells by IL-7-induced lymphokine-activated killer cells.

In order to assess the potential of interleukin 7 (IL-7) as an immunotherapeutic agent in human melanoma, we have evaluated the in vitro activity of IL-7-induced lymphokine-activated killer (LAK) cells from patients with advanced melanoma against allogeneic and autologous melanoma cells. Peripheral blood lymphocytes (PBLs) from 14 patients with stage III melanoma were isolated and incubated in the presence of 1,000 U ml-1 IL-7 and 100 U ml-1 IL-2 for comparison. LAK-cell activity was determined by a 24 h cytotoxicity assay using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The activity of IL-7-induced LAK cells against two allogeneic melanoma cell lines was 32.7% (+/- 17.9) against SK-Mel-37 and 38.1% (+/- 12.5) against SK-Mel-23 at an effector-to-target (E/T) ratio of 20:1. The activity of IL-2-induced LAK cells was significantly higher against SK-Mel-37 (78 +/- 24.6%) and against SK-Mel-23 (73.5 +/- 19.7%). IL-7 and suboptimal doses of IL-2 (10 U ml-1) were found to have a co-stimulatory on lymphocyte proliferation as well as on LAK activity. Against autologous melanoma cells, the activity of IL-7- and IL-2-induced LAK cells did not differ significantly (55.8 +/- 25.6% versus 68.7 +/- 21.7% respectively). In two patients, IL-7-induced LAK-cell activity against autologous melanoma cells exceeded even that of IL-2 significantly (67% vs 35% and 95% vs 82%). Levels of tumour necrosis factor alpha (TNF-alpha) in the supernatants of LAK-cell cultures generated by IL-7 were lower than those of IL-2-generated LAK-cell cultures. These results suggest that IL-7 is a potential alternative to immunotherapy with IL-2 in terms of efficacy and possible side-effects and encourages pilot studies with IL-7 in melanoma patients.

Adult↗
PubMed1994

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.

Antineoplastic Agents↗
PubMed1994

Interleukin-7 induces differential lymphokine-activated killer cell activity against human melanoma cells, keratinocytes, and endothelial cells.

To assess the potential role of interleukin (IL)-7 in immunotherapy of human malignant melanoma, we have examined the lymphokine-activated killer (LAK) cell sensitivity of four human melanoma cell lines against LAK cells generated by IL-7 or IL-2. Lysis was determined by a 24-h cytotoxicity test using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). All melanoma cell lines were susceptible to IL-7- and IL-2-generated LAK cells. The sensitivity of melanoma cells to IL-2-induced LAK cells was higher compared to IL-7-induced LAK cells. At an effector target ratio of 20:1, the lysis by IL-7-induced LAK cells ranged between 41% and 52%, whereas IL-2-induced lysis ranged between 80% and 94% (p < 0.01). IL-7-induced LAK cells, however, showed almost no cytotoxicity towards HaCat keratinocytes and human umbilical vein endothelial cells (HUVECs). Immunophenotyping revealed a higher expression of the tac antigen (CD 25) on IL-7-generated LAK cells, particularly those cells that were CD 56 negative or CD 3 positive compared to IL-2-induced LAK cells. In contrast, IL-2-generated LAK cells killed 62% of the HaCat keratinocytes and 60% of the HUVECs. Secretion of tumor necrosis factor-alpha into culture supernatants was significantly higher in IL-2-generated LAK cells compared to IL-7-stimulated LAK cells (p < 0.01), whereas TNF-alpha levels of IL-7-induced LAK cells were in the range of unstimulated lymphocytes. Because nonspecific cytotoxicity against other normal cells such as keratinocytes and endothelial cells contributes to the dose-limiting side effects of immunotherapy with IL-2, immunotherapy using IL-7 might be a better tolerated future alternative.

Cell Death↗
PubMed1994

Analysis of mast cell subpopulations (MCT, MCTC) in cutaneous inflammation using novel enzyme-histochemical staining techniques.

In order to gain insights into the dynamics of mast cell subpopulations in normal and diseased skin, a novel enzyme-histochemical double and triple staining method was employed that allowed the detection of metachromasia (toluidine blue) and the mast cell proteases tryptase and chymase within the same cell. Cryostat sections were used of skin biopsies from the following specimens: normal skin (N = 4), psoriasis (N = 13), atopic eczema (N = 7), lichen planus (N = 6), interferon alpha 2a injection sites (N = 1) of a leukemic infiltrate and corresponding normal skin of the same patient before and after treatment. (i) Equal numbers of tryptase- and chymase-positive mast cells (MCTC) were obtained in all normal and diseased specimens in papillary and reticular dermis, with threefold increases around appendages. (ii) Tryptase-positive mast cells (MCT) were absent in normal skin, but were markedly increased in a disease-specific pattern within the papillary dermis, the inflammatory infiltrate and around appendages. (iii) Marked increases of MCT were also noted at interferon injection sites within the leukemic infiltrate, but not in the normal skin of the same patient. These data suggest that disease-dependent mast cell dynamics involve only MCT in cutaneous inflammation and that MCT numbers are controlled by distinct, disease-specific local tissue factors.

Amino Acid Sequence↗
PubMed1993

Human mast cells produce IL-8.

Recruitment of neutrophils is a common feature in diseases that are associated with mast cell activation. The mechanisms that mediate neutrophil activation are not well understood. IL-8 is a recently described potent chemotactic factor that might be pathogenetically involved in this process. We therefore studied the human mast cell line HMCI and human skin mast cells for their ability to produce IL-8 using various stimuli. IL-8-mRNA was expressed in a stimulus- and time-dependent fashion as detected by Northern blot analysis with an IL-8-specific cDNA probe. The molecular mass of HMCI-derived IL-8 was determined to be about 8 kDa by immunoblot analysis. Immunoreactive and biologically active IL-8 protein was measured in the cell culture supernatants of HMCI cells by an ELISA and a chemotaxis assay, respectively. On immunoelectron microscopy of stimulated skin mast cells, IL-8 was found along cytoplasmatic membranes and in intracellular granules. Our data indicate that mast cells may contribute to neutrophil recruitment by secretion of IL-8.

Calcimycin↗
PubMed1993

Melanocytes as "sensory" and regulatory cells in the epidermis.

Epidermal melanocytes (MC) are pigment-producing and secretorily active cells of neural crest origin that communicate directly with multiple targets. Here, we propose that normal epidermal MC also are "sensory" and regulatory cells operating in the context of a regulatory network for the maintenance of human epidermal homeostasis. Altered regulatory MC functions may play a role in selected skin diseases, and racial pigmentation may affect cutaneous functions.

Cell Communication↗
PubMed1993

IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor.

Normal melanocytes require a number of exogenous growth factors in contrast to most metastatic malignant melanomas. This investigation demonstrates that endogenously produced human IL-8 can act as an important growth factor for human melanoma cells. In the present study, six out of eight human melanoma cell lines tested secrete IL-8 protein into the culture supernatant. In two of these IL-8-secreting melanoma cell lines, SK-MEL 13 and SK-MEL 23, we have determined the IL-8 requirement for their proliferative capacity. These melanoma cell lines produced significant amounts of bioactive IL-8 as measured by the ELISA technique. Secretion of human IL-8 was inducible by IL-1 and by PMA. Human IL-8-specific mRNA was already detected in unstimulated melanoma cells. In addition, human IL-8-R mRNA could be detected for the first time in human melanoma cells. Exposure of the two melanoma cell lines in vitro to antisense oligonucleotides targeted against two different sites of human IL-8 mRNA-inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 protein into culture supernatant in a dose dependent fashion. Effects were reversible either by removal of the oligomers or by addition of exogenous IL-8 protein. In contrast, exposure to IL-8 sense probes or oligonucleotides in sense or antisense orientation specific for IL-7, TGF-alpha, TGF-beta, and MGSA had no such effect. A monospecific immune serum and two IL-8-specific mAb were also capable of inhibiting melanoma cell proliferation in the same manner. These results provide strong evidence for an autocrine IL-8 synthesis and for an IL-8-dependent proliferation in a subgroup of human melanomas. Furthermore, they suggest that IL-8 may play a role not only in immunomodulation but also in melanoma progression and metastatic spread.

Base Sequence↗