Background: The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can also induce the expression of pro-angiogenic factors in fibroblasts postnatally. Misregulation of the Hh pathway has been implicated in a variety of different types of cancer, including pancreatic and small-cell lung cancer. Recently a putative antagonist of the pathway, Hedgehoginteracting protein (HIP), was identified as a Hh binding protein that is also a target of Hh signaling. We sought to clarify possible roles for HIP in angiogenesis and cancer.

We have studied the physical and functional linkages of heterologously expressed human 5-HT1A receptors to G protein alpha-subunits in HeLa and CHO-K1 cells. HeLa cells expressed immunoreactivity to G(i) proteins with an apparent rank order of G(i) alpha 3 (approximately 1 pmol/mg of protein) >> G(i) alpha 1 (approximately 0.1 pmol/mg) >> G(i) alpha 2 (< 0.02 pmol/mg), whereas CHO-K1 cells expressed immunoreactivity to G(i) alpha 2 (approximately 5 pmol/mg) >> G(i) alpha 3 (approximately 0.7 pmol/mg), but not to G(i) alpha 1. Both cell lines expressed large and small forms of Gs alpha, but neither expressed detectable G(o) alpha. Agonist-promotable physical coupling of the 5-HT1A receptor to G proteins was examined with high-affinity agonist binding and with co-immunoprecipitation using rabbit anti-receptor IgG fractions. Agonist treatment induced coupling of the 5-HT1A receptors to G proteins with an apparent rank order of G(i) alpha 3 > G(i) alpha 1, G(i) alpha 2 in HeLa cells and G(i) alpha 3 > G(i) alpha 2 in CHO-K1 cells. Agonist-promotable functional coupling of the 5-HT1A receptors to inhibition of adenylylcyclase was measured in membranes derived from HeLa and CHO-K1 cells expressing approximately 2.5-3 pmol of receptors/mg of protein by preincubation with antisera raised against the carboxyl termini of the G(i) protein alpha-subunits. A noteworthy difference between the two cell types was that antisera against the predominant G protein (G(i) alpha 2) were substantially more efficacious than G(i) alpha 3 antisera at blocking functional coupling to adenylylcyclase inhibition in CHO-K1 cells, whereas in HeLa cells, antisera against nonpredominant G proteins (G(i) alpha 1/G(i) alpha 2) were equally as effective as those against the predominant G protein (G(i) alpha 3). No physical or functional coupling of the 5-HT1A receptor to Gs alpha isoforms was detected in either cell line. These findings suggest that the 5-HT1A receptor can physically couple to multiple distinct G(i) proteins in mammalian cell membranes and that functional coupling to adenylylcyclase inhibition may be mediated by G(i) alpha 1, G(i) alpha 2, and G(i) alpha 3. One factor influencing the relative importance of those G proteins for 5-HT1A receptor-inhibited adenylylcyclase activity appears to be their-relative levels of expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Using an estrogen-inducible retroviral system, we demonstrate that oncogenic Raf-1 induces growth arrest and morphological changes in finite lifespan human mammary epithelial cells (HMEC). This arrest does not rely on expression of the cyclin-dependent kinase inhibitor (CKI) p16 INK4a , nor on changes in expression of the CKIs p21 Cip1 , p14 ARF , p27 Kip1 or p57 Kip2 . The Raf-induced arrest is independent of viral oncogene mediated inactivation of p53 and pRB, or c-myc overexpression. Flow cytometric analysis demonstrates that cells arrest in both G1 and G2. The Raf-induced arrest is mitigated or eliminated in some immortally transformed HMEC. Immortal HMEC that have both overcome replicative senescence and undergone the recently described conversion process maintain growth in the presence of transduced oncogenic Raf-1; they also gain EGF-independent growth and a low frequency of anchorage-independent growth. However, HMEC that have overcome replicative senescence but have not undergone conversion and HMEC immortalized by transduction with the catalytic subunit of telomerase, hTERT, remain severely growth arrested. These results indicate that the molecular mechanisms responsible for the Raf-1-induced growth arrest may vary among different finite lifespan cell types, and that in HMEC, this mechanism is altered during the conversion process, rather than as a direct consequence of overcoming senescence or expressing hTERT.

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