Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^