guanylyl-imidodiphosphate and Melanoma

Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.

Topics: Codon; DNA, Neoplasm; Exons; Female; Genes, ras; Guanylyl Imidodiphosphate; Humans; Male; Melanoma; Neoplasm Staging; Point Mutation; Polymerase Chain Reaction; Prognosis; ras Proteins; Retrospective Studies

The ability of a series of B16 melanoma clones to form experimental lung metastases in syngeneic mice has been shown to correlate positively with adenylate cyclase activity. (Sheppard et al, Int. J. Cancer 37 (1986) 713-722). To begin to identify the components of the adenylate cyclase complex that account for enhanced enzyme activity in highly metastatic tumor populations, cholate extracts containing the GTP-binding protein GS from B16 melanoma clones of different metastatic capacities were reconstituted with membranes prepared from S49 cyc-, a variant lymphoma cell line that lacks GS function. The results revealed that extracts from a highly metastatic B16 clone (F10-C23) reconstituted significantly greater adenylate cyclase activities in S49 cyc- membranes than parallel preparations from a B16 clone (F1-C29) of low metastatic capacity. The data suggest that aberrations in GS function may contribute to the heightened responsiveness of adenylate cyclase observed in B16 melanoma clones of increased metastatic potential.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Colforsin; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Melanoma; Mice; Molecular Weight; Neoplasm Metastasis

The regulation of adenylate cyclase in murine melanoma tumor cell clones with different metastatic capacities has been studied in intact cells and isolated membrane preparations. Analysis of the responses of intact cells from a number of B16 melanoma clones revealed that treatment with melanocyte-stimulating hormone (MSH) or the diterpene, forskolin, produced significantly greater accumulation of intracellular cyclic adenosine 3',5' monophosphate (cAMP) in strongly metastatic clones than in weakly metastatic tumor cell clones. In contrast, in isolated membranes from the same panel of clones, the extent of activation by forskolin but not by MSH correlated with metastatic capacity. Sodium fluoride and 5'-guanyl-beta-gamma-imidodiphosphate [Gpp(NH)p] also stimulated adenylate cyclase in isolated membranes but the extent of activation did not correlate with the metastatic behavior of the donor cells. A combination of forskolin and Gpp(NH)p proved to be a sensitive prospective indicator for identifying differences in the metastatic capabilities of individual B16 melanoma clones. Adenylate cyclase in membrane preparations from strongly metastatic B16 clones displayed synergistic activation but stimulation of the enzyme from weakly metastatic clones was less than additive. To test the generality of these findings, similar investigations were performed on B16-BL6 melanoma cells, a highly invasive subline of the B16 melanoma, and the K1735, an ultraviolet-light-induced murine melanoma arising in a different mouse strain (C3H). Consistent with their high metastatic potential, clones derived from the B16-BL6 melanoma displayed elevated levels of hormonally-stimulated adenylate cyclase, thereby confirming, for this tumor system, a close association between hormonal responsiveness and metastatic capacity. In contrast, K1735 melanoma cell clones exhibited significant interclonal variation in adenylate cyclase activity and metastatic performance, but no consistent relationship between the two traits was detected. Differences in the regulation and/or the intrinsic catalytic capacity of adenylate cyclase may account, at least in part, for the variation in hormonal responsiveness observed among B16 clones with distinct metastatic properties and suggest that cAMP-dependent molecular processes may be required for the expression of B16 melanoma experimental metastatic potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Clone Cells; Colforsin; Cyclic AMP; Fluorides; Guanylyl Imidodiphosphate; Kinetics; Lung Neoplasms; Melanocyte-Stimulating Hormones; Melanoma; Mice; Neoplasm Metastasis