In vivo and ex vivo MR imaging of slowly cycling melanoma cells.

Slowly cycling cells are believed to play a critical role in tumor progression and metastatic dissemination. The goal of this study was to develop a method for in vivo detection of slowly cycling cells. To distinguish these cells from more rapidly proliferating cells that constitute the vast majority of cells in tumors, we used the well-known effect of label dilution due to division of cells with normal cycle and retention of contrast agent in slowly dividing cells. To detect slowly cycling cells, melanoma cells were labeled with iron oxide particles. After labeling, we observed dilution of contrast agent in parallel with cell proliferation in the vast majority of normally cycling cells. A small and distinct subpopulation of iron-retaining cells was detected by flow cytometry after 20 days of in vitro proliferation. These iron-retaining cells exhibited high expression of a biological marker of slowly cycling cells, JARID1B. After implantation of labeled cells as xenografts into immunocompromised mice, iron-retaining cells were detected in vivo and ex vivo by magnetic resonance imaging that was confirmed by Prussian Blue staining. Magnetic resonance imaging detects not only iron retaining melanoma cells but also iron positive macrophages. Proposed method opens up opportunities to image subpopulation of melanoma cells, which is critical for continuous tumor growth.

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.

Melanoma cells and normal melanocytes share antigens recognized by HLA-A2-restricted cytotoxic T cell clones from melanoma patients.

HLA-A2-restricted, CD3+, CD8+, alpha/beta+ cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2+ melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2+, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous factors such as phorbol ester (O-tetradecanoyl phorbol 13-acetate [TPA]), epidermal growth factor, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2- allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on 21 targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal fibroblasts, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.

Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma-derived cell lines.

A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS. If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation-associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n = 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively) by array-based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n = 17, 13, 13 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1-36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2-24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS(wt/wt) tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations.

Specific antigen in serum of patients with colon carcinoma.

The binding of monoclonal antibody specific for colon carcinoma was inhibited by serum from patients with adenocarcinoma of the colon but not by serum from patients with other bowel diseases or from healthy volunteers. Of other malignancies studied, serum from two patients with gastric carcinoma and two patients with pancreatic carcinoma also inhibited the specific binding of monoclonal antibody. The levels of carcinoembryonic antigen in these serum samples were not correlated with their levels of binding inhibition. Such monoclonal antibodies may prove useful for the detection of colorectal carcinoma.

Melanocytes: from morphology to application.

Melanocytes in human skin are intricately regulated by keratinocytes and the surrounding stroma. The development of melanoma is thought to arise from disrupted melanocyte homeostasis. It is now known that microenvironment plays a major role in maintenance of cellular homeostasis and can contribute to tumor initiation and tumor progression. Historically, melanocyte studies have been performed in two-dimensional culture systems, and often with melanocytes cultured in the absence of keratinocytes. Here we present the biological basis for the use of organotypic, three-dimensional model systems in the study of melanoma, and highlight the features of the most utilized organotypic model systems.

Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Pathology and mass mortality of Pacific oysters, Crassostrea gigas (Thunberg), in 2005 at the East Frisian coast, Germany.

In 2005, Pacific oysters, Crassostrea gigas, were collected from May to September along the East Frisian coast and processed for histology. Because of mass mortalities in September, additional samples of moribund oysters and apparently healthy blue mussels, Mytilus edulis, were subjected to virological and ultrastructural investigation. The oysters displayed a variety of pathological conditions including viral gametocytic hypertrophy which is reported here for the first time from the German coast. Haemocyte aggregations in the digestive tract, in the intestinal mucosa and submucosa, in the mid-gut gland and in the ventricle of the heart were commonly observed at some stations. In association with mass mortalities, severe gill necrosis occurred which may have contributed to the high mortality rates. Total mortality rates of up to approximately 60% were seen. All size classes and thus age classes of oysters were affected, with highest mortality rates within the youngest age classes which had just reached sexual maturity (shell lengths <40 mm). The smallest dead oysters had shell lengths of 10 mm. The phenomenon was mainly restricted to C. gigas stocks in harbours, probably because of favourable conditions for infection, i.e. limited water exchange, less food availability, reduced oxygen content and higher pollution levels.

A slow-cycling subpopulation of melanoma cells with highly invasive properties.

Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates. Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized. Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties. We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs. Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.

Development of pemphigus vulgaris-like lesions in severe combined immunodeficiency disease mice reconstituted with lymphocytes from patients.

Pemphigus vulgaris is an autoimmune blistering disease that is induced by binding of antibodies to a 130/85-kD protein complex on epidermal keratinocytes. An in vivo experimental model of this disease was developed by reconstituting severe combined immunodeficiency (SCID) mice with 1-10 x 10(7) PBL from patients with naturally occurring pemphigus vulgaris. Of 49 reconstituted mice, 34 (69%) produced human IgG levels of > 0.1 mg/ml. Circulating anti-pemphigus antibodies were found in 20 of the 34 successfully reconstituted mice; 44% of these animals had deposits of human IgG in their own skin after it was traumatized by either heat or cold. Spontaneous pemphigus vulgaris-like blisters associated with human IgG deposits were rarely found in mouse skin. By contrast, allogeneic human skin grafted to 10 to 12 mice before reconstitution with patients' PBL developed pemphigus vulgaris-like lesions containing human IgG deposits. These results demonstrate that SCID mice can serve as a model of an antibody-mediated human autoimmune skin disease.

Human/severe combined immunodeficient mouse chimeras. An experimental in vivo model system to study the regulation of human endothelial cell-leukocyte adhesion molecules.

The ability of circulating white blood cells to enter inflamed tissues is mediated by specific cell adhesion molecules thought to be expressed in a programmed and sequential manner to form an "adhesion cascade." Because of the complexity of this process, it is becoming increasingly important to develop in vivo models. Two major problems have limited the utility of current animal models. The first is the inability of many of the antibodies developed against cell adhesion molecules in human cell culture models to cross-react in animals. The second is the uncertainty in extrapolating animal (particularly rodent) findings to humans. To circumvent these problems, full thickness human skin grafts were transplanted onto immunodeficient (severe combined immunodeficient) mice. After 4-6 wk, the transplanted skin grafts closely resembled normal skin histologically and maintained their human vasculature as determined by immunohistochemical staining with human-specific endothelial cell markers. Intradermal injection of tumor necrosis factor-alpha resulted in the reversible upregulation of the leukocyte-endothelial adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, and in an active inflammatory reaction with migration of murine leukocytes into cytokine-injected areas. These results indicate that the severe combined immunodeficient mouse/human skin transplant model provides a useful in vivo system in which to study human endothelium during the process of inflammation.

HLA-DR histocompatibility leukocyte antigens permit cultured human melanoma cells from early but not advanced disease to stimulate autologous lymphocytes.

HLA-DR histocompatibility antigens are commonly expressed by the melanocytes of melanoma and its precursors, but not by the melanocyte of normal skin. Further, the primary lesion of biologically early melanoma is commonly infiltrated with host T cells. Advanced disease is characterized by a paucity of such cells. To investigate the interaction of melanoma cells and autologous lymphocytes and its dependence on HLA-DR expression, we have established cell lines from biologically early (4 lines) and advanced disease (11 lines) and examined their capacity to stimulate blastogenesis of autologous T cells in vitro. Melanocytes from early disease expressed HLA-DR antigens and stimulated autologous T cells. Those from advanced disease, irrespective of DR expression, were nonstimulatory. To determine whether expression of DR was required for melanoma cells to be stimulatory, we first treated a stimulating cell line of DR3 allospecificity with anti-DR3-specific serum and demonstrated marked inhibition of its capacity to provoke blastogenesis. Next we used fluorescence-activated flow cytometry to sort a stimulating line heterogeneous for DR expression into DR-enriched and -depleted populations. When such cells were examined in the lymphocyte proliferation assay, their stimulatory capacity was proportional to their quantitative expression of HLA-DR. These studies indicate that cell lines may reflect important biological differences between early and advanced melanoma. HLA-DR expression may be an early event in neoplasia of melanocytes. These antigens are able to interact directly with autologous T cells; and their expression is necessary, but not sufficient, for melanoma cells to induce lymphocyte proliferation.

An engineered PAX3-KRAB transcriptional repressor inhibits the malignant phenotype of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene.

The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is dependent on the FKHR activation domain. We have fused the KRAB repression module to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.

Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a).

The tumor suppressor p16(INK4a) inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27(KIP1), and p21(WAF1/CIP1). Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14(ARF)/p53 tumor suppressor pathways.

Long-term 2007-2013 monitoring of reproductive disturbance in the dun sentinel Assiminea grayana with regard to polymeric materials pollution at the coast of Lower Saxony, North Sea, Germany.

During biological effect monitoring studies of endocrine active compounds with the snail Assiminea grayana in 2007-2013, reproductive disorders including atresia, transformation of capsule/albumen glands into prostates in females and ovotestis, transformation of prostates to capsule/albumen glands, disruption of spermatogenesis, and calcification of tubules in males, were encountered in several years. The search of sources of endocrine active substances was first directed to antifouling biocides from paint particles and extended to leaching compounds from polymeric materials. In contrast to the reference sites, most of the observed disorders occurred at a station near harbors and dockyards polluted with residues from antifouling paints and polymeric materials. Beside of investigations about the potential ingestion of polymer particles by the snails, further investigations of compounds of polymeric materials with endocrine potential should follow.

Towards the targeted therapy of melanoma.

Novel anti-cancer treatments use knowledge about the underlying biology of the tumor to find suitable molecular targets. The recent years have seen great advances in understanding the biology of melanoma. In the current review we discuss the most promising molecular targets for melanoma and suggest possible strategies for overcoming resistance.

Overexpression of IL-8 in the cornea induces ulcer formation in the SCID mouse.

AIMS: Although interleukin 8 (IL-8) is not produced in the normal cornea, it has been detected there in several pathological conditions. In this study, the direct effects of IL-8 overexpression on the cornea was examined. METHODS: The corneal surface of severe combined immunodeficiency mice was infected by the adenovirus vector encoding human IL-8 (IL-8/Ad5) and clinical and pathological changes were observed at various time points. RESULTS: Clinically, marked angiogenesis and ulcer formation in the cornea were observed by 12 hours and 24 hours, respectively. Histologically, prominent angiogenesis was observed in the corneal stroma at 12 hours. Cleft formation between the corneal epithelium and stroma, and neutrophil infiltration into the corneal stroma were seen at 16 hours. By 24 hours after the infection with IL-8/Ad5, a shallow ulcer was formed in the cornea. In contrast, infection with the control adenovirus carrying the beta galactosidase gene (LacZ) showed neither corneal ulceration nor neutrophil infiltration. Immunohistochemical analysis showed that infection with IL-8/Ad5 resulted in the production of IL-8 by corneal and conjunctival stromal cells. CONCLUSION: Our results indicate that IL-8 overexpression in corneal tissue causes ulcer formation in the cornea through chemoattraction of neutrophils, suggesting the aetiological role of IL-8 in some types of corneal ulcers.

Multiparameter evaluation of the expression in situ of normal and tumor-associated antigens in human colorectal carcinoma.

The immunoreactivity of a panel of monoclonal antibodies (MoAb) was studied in a series of patients with colorectal carcinomas to test the association of antigen expression with other parameters such as histopathologic stage, differentiation, and clinical outcome. Low-level binding to normal tissue and high-level binding to malignant tissue were observed with MoAb defining, respectively, a gastrointestinal cancer antigen (GICA), Leb (distal colon only), A, H type 2 antigen, X-like antigen, and the 200-kilodalton (Kd) protein of carcinoembryonic antigen (CEA). The degree of histologic differentiation correlated with the expression of Lea antigen, A, and Y haptens, whereas a progressive loss of these antigens coincided with loss of differentiation. Two undifferentiated carcinomas expressed only two, H type 2 antigen and a highly glycosylated protein of 20-50 Kd, of the 14 antigens investigated. An interesting, but not significant, association between Leb antigen expression and more extensive disease was found: Whereas 71% of Dukes C tumors were positive for Leb, only 48% of patients with Dukes A and B2 tumors showed the presence of Leb antigen. On the other hand, the presence of B72.3-defined antigen is significantly associated with an earlier stage of disease. Chi-square tests to assess the association of antigen positivity with disease recurrence indicated a significant binding association with tumor recurrence over a broad range of percent positive cells for two MoAb defining different determinants of GICA. Similar associations, but over a narrow range of positive cells, were found for H type 2 antigen and the 200-Kd protein of CEA.

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.