Experimental re-evaluation of the γ-ray energy and emission probability for the 159 keV transition in 238U following the α-decay of 242Pu.

Because of the very low specific activity of (242)Pu, the non-destructive assay of this isotope by means of conventional high-resolution gamma-spectrometry (HRGS) is possible only for Pu samples highly rich in (242)Pu. For bulk samples suffering from the gamma self-attenuation and self-fluorescence effects, the only practical choice for the quantitative analysis of (242)Pu is the weak γ-line emitted in the 159 keV transition of its α-decay daughter (238)U. A recent study revealed a significant disagreement between the (242)Pu mass values in a 99.72% enriched (242)PuO(2) sample as reported by HRGS and neutron coincidence counting. This fact motivated the present study on the experimental re-evaluation of the γ-emission probability for the 159 keV transition using a combination of α-, γ- and mass-spectrometry techniques. The obtained new emission probability P(2)=(2.20±0.08)10(-6) turned out to be ≈35% smaller than the currently adopted value. The study also suggested a new value E(2)=159.018±0.016 keV for the energy of the respective γ-ray.

Association of MC1R variants and host phenotypes with melanoma risk in CDKN2A mutation carriers: a GenoMEL study.

BACKGROUND: Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma. Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process. However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited. METHODS: We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium. In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers. These associations were estimated and tested using generalized estimating equations. All statistical tests were two-sided. RESULTS: Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10(-6) ≤ P ≤ .0007). A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants. The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; P(trend) = 1.86 × 10(-8)). The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10(-6) ≤ P ≤ .02). CONCLUSION: Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers. This joint association may have important consequences for risk assessments in familial settings.

CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations.

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

A versatile method for the removal of melanin from ribonucleic acids in melanocytic cells.

Melanin pigments often co-purify during preparation of nucleic acids from cells or tissues of melanocytic origin. Contaminating melanin can severely impede subsequent analyses of RNA. We attempted to eliminate melanin in RNA preparations using selected gel matrices. We show here that co-purified melanin pigments can be largely eliminated from RNA samples after passing through polyacrylamide-based beads (Bio-Gel P-60). After isolation from the pigment-containing cells or tissues, RNA was subsequently processed through batch or column purification under acidic pH conditions. The resulting RNA was devoid of contaminating melanin pigments and amenable to molecular reactions such as polymerase chain reaction and cDNA synthesis by reverse transcriptase. Although the process results in some loss of input RNA, this purification procedure is simple, robust and can easily be adopted in any laboratory for the molecular analysis of RNA that requires removal of melanin contamination.

Isolation of the melanoma-associated antigen p23 using antibody phage display.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.

E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.

In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation.

Human melanoma progression in skin reconstructs : biological significance of bFGF.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

Progression-related expression of beta3 integrin in melanomas and nevi.

The expression of the beta3 integrin subunit was investigated in 130 fixed, paraffin-embedded specimens of human melanomas and nevi using two different monoclonal antibodies. Expression was not observed in melanocytes and was absent or low in most nevi. In primary melanomas, expression was absent or low in the nontumorigenic radial growth phase, which includes the classes of in situ and microinvasive melanomas. In contrast, expression was high in the tumorigenic or vertical growth phase compartment of many primary melanomas and in most metastatic melanomas. Expression patterns were similar with the two antibodies, SSA6 and SAP, and was membrane-related as well as cytoplasmically expressed. In those nevi that reacted focally, the reactivity tended to occur in the dermal component of neurotized nevi, and in Spitz nevi, where the reactivity was stronger and more diffuse. A few dysplastic nevi showed focal reactivity of the junctional component. These results are consistent with tumor progression-related expression of the beta3 integrin, which is expressed in melanocytic tumors as the alphavbeta3 integrin, having affinity for matrix molecules, including vitronectin and fibronectin. In all melanomas, and in the subset of tumorigenic vertical growth phase melanomas, expression increased with thickness (P < .01). For this reason, and because ligation of this integrin has been shown in vitro to have several properties that may be related to the malignant phenotype, it is likely that expression of this marker may have prognostic value. However, because of its consistent and strong expression in Spitz nevi, the diagnostic utility of this marker will likely be limited.

Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Proto-oncogene c-kit expression in malignant melanoma: protein loss with tumor progression.

The c-kit gene encodes a transmembrane receptor that has tyrosine kinase activity. c-kit plays a role in hematopoiesis, gametogenesis, and melanogenesis. c-kit is found in melanocytes, and there is evidence that expression is lost in melanoma. We studied 85 melanocytic lesions for c-kit by immunohistochemical techniques using a monoclonal antibody. The lesions included banal nevi, junctional and compound nevi with melanocytic dysplasia, nontumorigenic radial growth phase melanoma, tumorigenic vertical growth phase melanoma, and metastatic melanoma. We found intense membrane staining in normal melanocytes and mast cells. Staining in compound nevi was strongest in junctional and superficial dermal components, whereas dermal nevi showed weak reactivity. Dysplastic nevi stained strongly, particularly in junctional cells. In melanoma, strong reactivity was most prominent in radial growth phase disease, but there was little or no staining in vertical growth phase and metastatic melanomas. In summary, c-kit protein is expressed in normal melanocytes, benign nevi, dysplastic nevi and nontumorigenic melanoma, but expression is lost in tumorigenic primary melanomas and metastases. The role of c-kit loss in advanced melanoma requires additional investigation.

A model system for detection and isolation of a tumor cell surface antigen using antibody phage display.

To establish a screening procedure for tumor cell-surface reactive Fabs, we used a model antigen/antibody system including the epidermal growth factor receptor (EGF-R) and the anti-EGF-R monoclonal antibody 425. The 425 Fab was displayed on the surface of M13 filamentous phage. In a screening assay for 425 phage binding to tumor cell surfaces, biotinylated 425-phage bound specifically to EGF-R-positive A431 epidermoid carcinoma cells and not to K562 non-expressor erythroleukemia cells. With a model library, the sensitivity of phage enrichment by phage binding to cell surfaces was one 425-phage in 20,000 unrelated phages after 4 rounds of panning on A431 cells. In a phage tissue screening assay, 425-phage, but not unrelated phage, bound specifically to melanoma cells expressing EGF-R. Epitope and idiotope specificity of 425-phage was demonstrated in phage competition assays, using as targets A431 cells and anti-idiotypic antibodies to monoclonal antibody 425, respectively. Finally, the EGF-R protein was directly isolated from A431 cell extracts, using biotinylated 425-phage. The data obtained with the 425 model library system demonstrate the usefulness of antibody phage display for the rapid identification and isolation of tumor or other disease-related cell surface antigens.

Combinatorial antibodies against human malignant melanoma.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for > 7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10(8) phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a > 10(6)-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.

Neoplastic progression and prognosis in melanoma.

Most melanomas evolve through an initial stage known as radial growth phase (RGP), encompassing in situ and microinvasive malignancies in which the probability of cure approaches 100%. At the present time, despite a shift toward earlier recognition of melanoma, by the time of diagnosis roughly 70% of melanomas have evolved to a point, known as vertical growth phase (VGP) or tumorigenic melanoma, at which cure is not certain, and prognosis depends upon certain attributes of the neoplasm and the host. Attempts have been made to assemble these attributes into prognostic models to permit estimation of the probability of cure for individuals and for groups of patients. Attributes that have been identified as independent prognostic variables include thickness of the primary neoplasm, the numbers of mitotic figures, and the presence of tumor-infiltrating lymphocytes (TIL). Other biologically important prognostic variables are on the horizon, and some will likely be based on molecules (markers) expressed on neoplastic cells that show functional significance in mechanisms of metastasis.

Melanoma-associated expression of transforming growth factor-beta isoforms.

Melanocytic neoplasia is characterized by the aberrant overproduction of multiple cytokines in vitro. However, it is largely unknown how cytokine expression relates to melanoma progression in vivo. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine commonly produced by cultured melanoma cells. The in situ expression of all three TGF-beta isoforms (TGF-beta1, -2, and -3) was determined immunohistochemically in melanocytes and in 51 melanocytic lesions using isoform-specific antibodies. Significant linear trends of expression were observed from melanocytes through nevi and primary and metastatic melanomas for all three isoforms. TGF-beta1 was expressed by some melanocytes and almost uniformly by nevi and melanomas. TGF-beta2 and -3 were not expressed in normal melanocytes but were expressed in nevi and early and advanced primary (radial and vertical growth phase) and metastatic melanomas in a tumor-progression-related manner. TGF-beta2 was heterogeneously expressed in advanced primary and metastatic melanomas, whereas TGF-beta3 was uniformly and highly expressed in these lesions. Thus, expression of TGF-beta isoforms in melanocytes and melanocytic lesions is heterogeneous and related to tumor progression, and expression of TGF-beta2 and TGF-beta3 commences at critical junctures during progression of nevi to primary melanomas.