The metastasis suppressor KISS1 lacks antimetastatic activity in the C8161.9 xenograft model of melanoma.

The objective of this study was to use the established xenograft model of human melanoma (C8161.9) to test a pharmacological approach to the effect of the metastasis suppressor KISS1. A KISS1 analog was used to inhibit the metastatic development of C8161.9 cells in nude mice. Further experiments were performed to test the validity of the C8161.9 model and test the connection between KISS1 expression and loss of metastatic potential. New clones of C8161.9 cells were obtained, with or without KISS1 expression, and were tested for metastasis formation. The absence of benefit in survival with the KISS1 analog compared with PBS prompted us to revisit the C8161.9 model. We found that the cells expressing KISS1, used in the previous study and obtained by transfection and single-cell cloning, were defective for both formation of orthotopic tumors and metastases. In mixing experiments, these cells could not suppress orthotopic tumor growth of KISS1-negative C8161.9 cells, suggesting that the suppression of metastasis by C8161.9-KISS1 cells may be intrinsic to the selected clone rather than related to KISS1 expression. Isolation of clones from parental C8161.9 cells in soft agar yielded cell populations that phenotypically and genotypically mimicked the KISS1-positive clone. In addition, new clones expressing KISS1 did not show any decrease in metastatic growth. These data demonstrate the heterogeneity of cell types in the C8161.9 cell line and the high risk of artifact linked to single-cell selection. A different xenograft model will be necessary to evaluate the use of KISS1 analogs as antimetastatic therapy

Heat Shock Response in CHO Mammalian Cells Is Controlled by a Nonlinear Stochastic Process

In many biological systems, the interactions that describe the coupling between different units in a genetic network are nonlinear and stochastic. We study the interplay between stochasticity and nonlinearity using the responses of Chinese hamster ovary (CHO) mammalian cells to different temperature shocks. The experimental data show that the mean value response of a cell population can be described by a mathematical expression (empirical law) which is valid for a large range of heat shock conditions. A nonlinear stochastic theoretical model was developed that explains the empirical law for the mean response. Moreover, the theoretical model predicts a specific biological probability distribution of responses for a cell population. The prediction was experimentally confirmed by measurements at the single-cell level. The computational approach can be used to study other nonlinear stochastic biological phenomena

Activation of Rho and Rho-Associated Kinase by GPR54 and KiSS1 Metastasis Suppressor Gene Product Induces Changes of Cell Morphology and Contributes to Apoptosis

ABSTRACT The mechanism of action of the metastasis suppressor KiSS1 and its receptor GPR54 is still incompletely characterized. Although the loss of KiSS1 expression by tumor cells has been associated with a metastatic phenotype, the nature of the cellular target of the secreted kisspeptins is unknown. Although an autocrine model of action has been generally assumed, metastasis suppression by KiSS1 has also been shown in cells that do not express GPR54, suggesting a paracrine mechanism in which kisspeptins affect cells in the metastatic niche. Activation of GPR54 was shown to inhibit cell motility and invasion of tumor cells, induce the formation of stress fibers, and reduce the expression of matrix metalloproteinase 9. We showed previously that the activation of GPR54 by kisspeptin-10 suppressed CXCR4-mediated chemotaxis in response to stromal cell-derived factor 1/CXCL12 and abolished the phosphorylation of Akt by CXCR4. We also demonstrated that activation of GPR54 inhibited Akt phosphorylation after the activation of epidermal growth factor receptor and the insulin receptor and triggered apoptosis in epithelial and lymphoid cell lines through a mechanism involving extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. We show here that the activation of GPR54 induced immediate and profound changes of cell morphology, including cytoplasmic condensation and formation of unpolarized plasma membrane protrusions. These events were dependent on Rho and Rho-Associated Kinase (ROCK) activation. The activation of ROCK also contributed to GPR54-mediated apoptosis in 293 cells, and its effect was additive to and independent of ERK activation. These results suggest that RhoA and ROCK are additional key components of the antimetastatic effect of kisspeptins. KiSS1 was identified as a metastasis suppressor gene in human malignant melanoma cells KiSS1 was originally identified in experiments in which the transfer of human chromosome 6 to metastatic melanoma cells suppressed metastasis in a mouse xenograft model in mouse This work was supported by the Georgia Cancer Coalition

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes.

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells