Uta B. Hofmann

Expression of integrin αvβ3 correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo

Activation of matrix metalloproteinase‐2 (MMP‐2) is mediated by binding to the complex of membrane‐type matrix metalloproteinase‐1 (MT1‐MMP) with tissue inhibitor of MMP‐2 (TIMP‐2) on the cell surface. Binding of MMP‐2 to integrin αvβ3 has been implicated in presenting activated MMP‐2 on the cell surface of invasive cells, but interactions with the MT1‐MMP‐TIMP‐2 system have not been considered. Therefore, we studied the expression and interaction of MT1‐MMP, MMP‐2 and TIMP‐2 in the αvβ3‐negative melanoma cell line BLM and in its β3‐transfected, αvβ3‐expressing counterpart BLM‐β3, both on cell lines and in xenografts. Total expression levels of MMP‐2, MT1‐MMP and TIMP‐2 did not differ markedly between the αvβ3‐negative and αvβ3‐positive cells. Remarkable differences, however, exist in the presence of active MMP‐2 and MT1‐MMP. Zymography on cell lysates revealed that active MMP‐2 was restricted to αvβ3‐positive cell line and clearly accumulated in xenografts derived from the BLM‐β3 cells, confirming the relevance of this integrin for MMP‐2 function. Western blotting of cell lysates showed that processing of proMT1‐MMP to the activated form was enhanced in BLM‐β3. The ratio of active and inactive MT1‐MMP was 3‐fold higher in the β3‐transfectants. Immunofluorescence double‐labeling followed by confocal laser microscopy showed co‐localization of MT1‐MMP and αvβ3 on BLM‐β3 cells. In xenografts from BLM‐β3 cells, active MT1‐MMP was markedly increased. Our results demonstrate that expression of αvβ3 in cell lines and xenografts was accompanied by an accumulation of active MT1‐MMP and MMP‐2. Furthermore, MT1‐MMP and αvβ3 are co‐localized on the cell membrane of tumor cells. These findings suggest that activated MT1‐MMP co‐localized with αvβ3 may be involved in activation of αvβ3‐bound MMP‐2. Int. J. Cancer 87:12–19, 2000.

Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis.

Narrow-band UVB311 nm vs. broad-band UVB therapy in combination with topical calcipotriol vs. placebo in vitiligo

Background  Recently, it has been shown that UVB phototherapy may be more effective than UVA in the treatment of vitiligo. Currently, however, no studies have compared the efficacy of UVB311 nm and broad‐band UVB therapy. Calcipotriol has recently been reported to be effective adjunctive treatment for vitiligo, enhancing the efficacy of 8‐methoxypsoralen plus UVA (PUVA) therapy.

Overexpression of the KIT/SCF in Uveal Melanoma Does Not Translate into Clinical Efficacy of Imatinib Mesylate

Purpose: Recently, gene amplification and overexpression of KIT as well as activating mutations in the KIT gene have been described to occur in certain subsets of melanoma. These findings suggest KIT as a potential target for therapy with imatinib mesylate in these melanomas. To date, data on the KIT status in uveal melanoma (UM) is limited. Experimental Design: We analyzed the expression of the KIT protein (CD117, c-kit) and its ligand, stem cell factor (SCF), in primary and metastatic UM. Results: By immunohistochemistry, SCF-positive tumor cells (>90%) were detectable in 43% of primary UM and in 58% of UM metastases. Strong expression of KIT (>90%) in tumor cells was present in 55% of primary UM and in 76% of UM metastases. This overexpression of both KIT and SCF suggests the clinical application of imatinib mesylate in metastatic UM. This notion was tested in a clinical study using Simons two-stage design. Patients received imatinib (600 mg p.o. daily) until progress or unacceptable toxicities. The trial did not enter stage II as no objective response was observed in the first group. This observation prompted further molecular analysis, which revealed no mutations in the genomic sequence of KIT in exons 11, 13, 17, and 18. Moreover, the mitogen-activated protein kinase pathway was not activated in any of the tumors as measured by ERK phosphorylation. Conclusions: These results show the lack of clinical effectiveness of imatinib in UM, which was originally anticipated based on the high levels of KIT and SCF expression.

CD147 Impacts Angiogenesis and Metastasis Formation

CD147 is highly expressed on many tumor cells; its role for tumor invasiveness and metastasis has been deduced from its capacity to induce MMPs, i.e., MMP-1, -2, -3, and -9. However, in the murine B16 melanoma model, MMP-2/-9 expression occurs independent of CD147. To scrutinize the impact of CD147 on metastasis formation and angiogenesis in this model, CD147 was stably knocked down in B16 cells. This silencing of CD147 expression resulted in a reduced capability of the tumor cells to metastasize to the draining lymph nodes. Notably, the CD147 knock down caused a decreased VEGF expression in vivo accompanied by reduced blood vessel formation. Thus, in the B16 melanoma model, CD147 promotes metastasis formation by induction of angiogenesis in an MMP independent manner.

Stromal cells as the major source for matrix metalloproteinase-2 in cutaneous melanoma

Matrix metalloproteinases (MMPs) are essential for tumor progression, invasion and metastases formation. Expression of these proteinases is not only restricted to the tumor cells themselves, but also is found in normal stromal cells. Moreover, immunohistochemistry suggests stromal cells as the major source. To scrutinize this hypothesis we established a slowly growing, syngeneic tumor model using the B16-melanoma cell line B78D14. In vitro analysis demonstrated that B78D14 cells secreted MMP-2, MT1-MMP, and to a lesser degree MMP-9; in addition they expressed both MT1-MMP and EMMPRIN on their surface. In subcutaneous (s.c.) tumors of these cells MMP-2 expression was predominantly present at the tumor-stroma border indicating stromal cells as primary source for this protease in vivo. Indeed, double staining experiments and in situ zymography confirmed that tumor adjacent stromal cells at the invasive front expressed MMP-2 and only at this site activated MMP-2 was detectable. Notably, in an experimental pulmonary metastases model neither tumor nor stromal cells expressed MMP-2, suggesting that the capacity of stromal cells is largely dependent on the surrounding microenvironment.

Simultaneous Onset of Segmental Vitiligo and a Halo Surrounding a Congenital Melanocytic Naevus

Unlike in common melanocytic naevi, an acquired leukoderma (halo) surrounding a congenital melanocytic naevus is a rare phenomenon. A 6-year-old boy developed a depigmentation around a congenital melanocytic naevus on the right thigh. Simultaneously, segmental vitiligo appeared on the thigh, lower abdomen and buttock of the same side with sharp midline demarcation. Examination for associated autoimmune diseases proved negative. The simultaneous occurrence of a halo phenomenon around a congenital melanocytic naevus and segmental vitiligo, as well as identical histological and immunohistological findings in both pigmented lesions, suggest shared immunological mechanisms.

Congenital unilateral speckled lentiginous blue nevi with asymmetric spinal muscular atrophy

We describe a 30-year-old man presenting with congenital widespread unilateral hyperpigmented areas on the right side of the face and limbs that were studded with multiple blue nevi. There was nevus of Ota-like scleral involvement of the right eye. Histologic examination showed common blue nevi, one in association with lentigo. For this unusual type of dermal melanocytosis we propose the term, congenital unilateral speckled lentiginious blue nevi. In addition, the patient had monomelic spinal muscular atrophy of his right shoulder girdle and arm.

Identification and characterization of survivin‐derived H‐2Kb‐restricted CTL epitopes

Survivin is overexpressed in several malignancies and in tumor‐associated endothelium making it an attractive target for therapeutic cytotoxic T‐cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence for potential binding Kb‐restricted octamer peptide epitopes. Two epitopes, which bind strongly to Kb, were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes. The natural processing and presentation of these epitopes by tumor cells was evident by the killing of murine melanoma cells by vaccination‐induced T cells. Subcutaneous challenge with syngeneic melanoma demonstrated the protective immunity of this vaccination. Notably, analysis of the vessel density in subcutaneous tumors revealed that survivin‐specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two Kb‐restricted peptide epitopes derived from the murine survivin protein; moreover, survivin‐specific vaccination not only resulted in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin‐directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model.

Streifen- und wirbelförmige nävoide Hypermelanose : Fallbeschreibung und Literaturübersicht

ZusammenfassungWir diagnostizierten bei einem 7jährigen Patienten eine streifen- und wirbelförmige nävoide Hypermelanose (SWNH). Die wesentlichen Kriterien dieser 1988 beschriebenen Entität sind lineare, oft retikuläre Hyperpigmentierungen entlang der Blaschko-Linien ohne entzündliches Vorstadium und ohne Atrophie der Läsionen. Die Erkrankung manifestiert sich meist innerhalb der ersten 2 Lebensjahre. Histologisch ist eine verstärkte Pigmentierung des Stratum basale mit prominenten Melanozyten zu finden. Im Gegensatz zu früheren Beobachtungen fanden sich bei unserem Patienten einige Melanophagen im oberen Korium im Sinne einer Pigmentinkontinenz. In wenigen Fällen wurden assoziierte Mißbildungen mitgeteilt, fanden sich im vorgestellten Fall jedoch nicht. Anhand dieses Fallberichtes möchten wir das klinische Bild, die diagnostischen Kriterien und die Differentialdiagnosen dieser seltenen Entität darstellen. Soweit uns bekannt, stellt unser Patient den ersten Fall einer SWNH in der deutschsprachigen Literatur dar.SummaryA 7 year old boy presented with linear and whorled nevoid hypermelanosis (LWNH). This entity, delineated in 1988, is characterized by streaked or whorled, frequently reticulate hyperpigmented lesions following the lines of Blaschko without preceding inflammation and without atrophy. The age of onset is usually within the first 2 years of life. Histologically, there is a mild basal cell hyperpigmentation with prominent melanocytes. In contrast to earlier reports, we observed some melanophages in the upper dermis. Associated abnormalities have been reported in a few patients, but were absent in our case.We present the clinical features, diagnostic criteria and differential diagnosis of this rare entity. Apparently, our patient represents the first case of LWNH in the German literature.