Cytoplasm

Fig. 1 Sonic Hedgehog pathway. SHH acts on the receptor complex of Ptch and Smo to inhibit repression of Smo by Ptch. Smo then signal activates gli intracellularly, resulting in downstream transcription of target genes. (View this art in color at www.dekker.com.)

et al.,[12] noting that M-Shh-N and Ptch colocalize in subcellular vesicles, suggested that internalization of M-Shh-N is mediated by Ptch. At present it is unclear whether Ptch eventually separates from M-Shh-N, with M-Shh-N being directed to lysosomes for degradation and Ptch returning to cell surface. It has been suggested that Ptch and Smo shuttle oppositely between the plasma membrane and cytoplasmic vesicles in response to M-Hh-N ligand.

In the absence of M-Hh-N binding, Ptch may be found principally at the cell membrane. At the same time, Smo may occupy intracellular vesicles. The binding of M-Hh-N to Ptch triggers a dynamin-mediated internalization into endosomes[13] and this results, indirectly, in the migration of vesicular Smo to the cell membrane.

Caveolae and caveolin-1 protein, which coats caveolae, may play a role in M-Hh-N uptake by Ptch.[14] However, only Ptch interacts specifically with caveolin-1 whereas Smo does not.

Ptch mediates the posttranslational modification of the C-terminal domain of Smo. Modification may include potential enzymatic candidates such as a phosphatase, Protein kinase A, or cyclin Bl that alter the phosphorylation status of Smo, affecting its stability, activity, and subcellular localization.1-15-1 When Smo moves to the cell surface, hyperphosphorylation takes place, a putative Smo kinase increasing its activity. How closely linked phos-phorylation of Smo is to its transport to the cell surface is not known at present. Unstimulated Ptch may promote dephosphorylation of Smo, a putative Smo phosphorylase decreasing Smo's activity.

Denef et al.[16] provided some evidence that Ptch and Smo do not interact physically in transducing M-Hh-N signals. They suggested that the regulatory interaction between Ptch and Smo need not be stoichiometric. It has been found[5] that Ptch and Smo are not significantly associated together within responding cells. They demonstrated that Ptch, unbounded by M-Hh-N, acts substoi-chiometrically to suppress activity of Smo. Extremely low levels of Ptch were sufficient to substantially reduce Smo activity. For example, a Ptch:Smo ratio of 1:45 suppressed nearly 80% of Smo activity. They suggested that in the absence of M-Hh-N, Ptch may translocate a small molecule across the lipid bilayer that regulates the activity state of Smo.

In contrast, Incardona et al.[6] provided some evidence that supported a model in which colocalization of Ptch and Smo occurs in the absence of M-Shh-N. On M-Shh-N binding to Ptch, both Ptch and Smo may undergo simultaneous transport into the endocytic pathway where Smo is subsequently segregated from the Ptch/M-Shh-N complex, which is destined for lysosomal degradation. Possibly, late endosomal sorting may control the generation of active Smo.

Whether Ptch eventually separates from M-Shh-N, with M-Shh-N being directed to lysosomes for degradation and with Ptch returning to the cell surface, is unclear at present.

To date, NBCS has not been reported with mutations in PTCH2, SHH, SMO, or GUI, but there are reasons for believing that such mutations may be found. PTCH2 has been reported with a mutation in a medulloblastoma, whereas medulloblastoma is a feature of NBCS in about 3-5% of cases. A disputed mutation in SHH has been reported in one isolated BCC. Furthermore, BCCs and skeletal malformations are reported in hypersonic trans-genic mice. Mutations in SMO have been reported in isolated basal cell carcinomas, and expression of GLIl has been found in almost all isolated BCCs studied.

Target genes for this disorder include Ptch (PTCH), the Wnt (WNT) family, and the bone morphogenetic proteins (BMPs) of the TGFp superfamily. These are essential for normal embryonic development and differentiation of many adult tissues. Experiments in mice, chicks, and flies have shown that modification of genes or exposure to teratogens causes interesting and similar changes to reported human phenotypes. For example,

Ptch +/ — in mice results in skeletal defects and neoplasms, but changes can be inhibited by cyclopamine. [This plant steroidal alkaloid induces cyclopia in vertebrate embryos and has been shown to act by inhibiting the cellular response to the Shh signal (see below)].

Ptch — /— in mice is lethal, producing overgrown and open neural tubes. Ptch1130X in flies causes C-terminal truncation and abolishes Smo repression, but not Hh binding. PtchAloop2 in chicks cannot bind Shh ligand. Ptchster°l sensor mutations in flies abolishes Smo repression, but not HH binding.

Taipale et al.[17] showed that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. The substance was discovered through epidemiological investigations of cyclopia in sheep in the Western United States. Cyclopia was found to result when pregnant females ate the lily Veratrum californicum. The ''active'' ingredient was named ''cyclopamine.'' Although cyclopamine has no apparent effect on adult animals, it consistently leads to severe holoprosencephaly in developing embryos. Because this condition can be caused by either mutations in human hedgehog or exposure to cyclopamine, it was proposed that cyclopamine suppresses Hedgehog. Taipale et al.[17] demonstrated that cyclopamine acts by affecting the balance between active and inactive forms of

Smoothened and may therefore have a role in treating BCCs and BCNS.

As epidemiological studies have shown, adult sheep tolerate cyclopamine well, and the results are encouraging. They do not directly address the question of whether switching off the Hedgehog pathway will cure basal cell carcinomas or prevent BCNS. BCCs, like most other human cancers, have mutations in many genes, and it is probably the accumulation of mutations that results in malignancy. Perhaps the Hedgehog pathway must be activated to kick-start tumor development, but may not need to be kept activated to maintain a fully malignant state. Indeed, in naturally occurring BCCs, shutting off this pathway might be too little, too late! Treating basal cell carcinomas with cyclopamine might be expected to suppress the development of neoplastic cells and perhaps limit growth, but not automatically kill them. There is some early evidence that tumor cells that have been suppressed in this way might undergo programmed cell death or be eradicated by the body's natural defenses. If not, then tumors treated with cyclopamine may eventually reappear when the drug is stopped.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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