Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.

The correct patterning of vertebrate skeletal elements is controlled by inductive interactions. Two vertebrate hedgehog proteins, Sonic hedgehog and Indian hedgehog, have been implicated in skeletal development. During somite differentiation and limb development, Sonic hedgehog functions as an inductive signal from the notochord, floor plate and zone of polarizing activity. Later in skeletogenesis, Indian hedgehog functions as a regulator of chondrogenesis during endochondral ossification. The vertebrate Gli zinc finger proteins are putative transcription factors that respond to Hedgehog signaling. In Drosophila, the Gli homolog cubitus interruptus is required for the activation of hedgehog targets and also functions as a repressor of hedgehog expression. We show here that Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 (C.-c. Hui and A. L. Joyner (1993). Nature Genet. 3, 241-246) mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development. The role of Gli2 and Gli3 in Hedgehog signaling during skeletal development is discussed.

Carbonoyloxy analogs of the anti-metastatic drug swainsonine. Activation in tumor cells by esterases.

Swainsonine (SW), a plant alkaloid and inhibitor of alpha-mannosidases, has been shown to inhibit N-linked oligosaccharide processing and to block tumor cell metastasis in mice. In this study, a series of SW analogs were chemically synthesized and compared for inhibition of complex-type N-linked oligosaccharide processing in cultured MDAY-D2 tumor cells, for inhibition of alpha-mannosidases in vitro, and for stimulation of bone marrow proliferation in vivo. Carbonoyloxy substitutions at the 2 and 8 carbons of SW reduced inhibitor activity by 2-3 orders of magnitude for Jack Bean and MDAY-D2 tumor cell lysosomal alpha-mannosidases in vitro. However, 2-p-nitrobenzoyloxy-, 2-octanoyloxy- and 2-butanoyloxy-derivatives of SW retained full activity as inhibitors of Golgi oligosaccharide processing in viable MDAY-D2 tumor cells. Inhibition of oligosaccharide processing was reduced by the esterase inhibitor diethyl p-nitrophenyl phosphate, suggesting that although 2-p-nitrobenzoyloxy-SW, 2-octanoyloxy-SW and 2-butanoyloxy-SW are relatively poor inhibitors of alpha-mannosidases in vitro, the compounds enter cells at a rate comparable to that of SW, and are converted to SW by cellular esterases. The more lipophilic esters, 2-benzoyloxy-SW, 2-toluoyloxy-SW, 8-palmitoyloxy-SW and 8-myristinoyloxy-SW, showed IC50 values at least 10 times higher for inhibition of Golgi oligosaccharide processing, probably due to less efficient entry of the compounds into tumor cells. The anti-metastatic activities of SW and two analogs were tested and shown to correlate with the IC50 values for inhibition of Golgi oligosaccharide processing in cultured tumor cells. In vivo, SW and the analogs were administered intraperitoneally to mice and found to have comparable activities as stimulators of bone marrow cell proliferation. Carbonoyloxy substitutions at the 2- or 8-position of SW with other chemical groups may lead to new drugs with improved pharmacokinetics and anti-cancer activity.