Identification of a tetrameric hedgehog signaling complex.

Hedgehog (Hh) signal transduction requires a large cytoplasmic multi-protein complex that binds microtubules in an Hh-dependent manner. Here, we show that three members of this complex, Costal2 (Cos2), Fused (Fu), and Cubitus interruptus (Ci), bind each other directly to form a trimeric complex. We demonstrate that this trimeric signaling complex exists in Drosophila lacking Suppressor of Fused (Su(fu)), an extragenic suppressor of fu, indicating that Su(fu) is not required for the formation, or apparently function, of the Hh signaling complex. However, we subsequently show that Su(fu), although not a requisite component of this complex, does form a tetrameric complex with Fu, Cos2, and Ci. This additional Su(fu)-containing Hh signaling complex does not appear to be enriched on microtubules. Additionally, we demonstrate that in response to Hh Ci accumulates in the nucleus without its various cytoplasmic binding partners, including Su(fu). We discuss a model in which Su(fu) and Cos2 each bind to Fu and Ci to exert some redundant effect on Ci such as cytoplasmic retention. This model is consistent with genetic data demonstrating that Su(fu) is not required for Hh signal transduction proper and with the elaborate genetic interactions observed among Su(fu), fu, cos2, and ci.

Characterization of the intracellular distribution and binding in human adenocarcinoma cells of N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), a photoaffinity analogue of the antitumor diarylsulfonylurea sulofenur.

A photoactivatable diarylsulfonylurea, N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), has been examined as a potential probe to elucidate the intracellular distribution and binding of antitumor diarylsulfonylureas. Our results demonstrated that against the human colon adenocarcinoma cell line GC3/c1, LY219703 is a more potent cytotoxic agent than N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (Sulofenur; ISCU), whereas a subline selected for resistance to ISCU was cross-resistant to LY219703, suggesting a similar mechanism of action or resistance. Cellular pharmacology studies showed that [3H]LY219703 concentrated in cells, and that its concentrative accumulation could be inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that it was similar to other antitumor diarylsulfonylurea (DSU) drugs examined. Accumulation of [3H]LY219703 in cells was progressively decreased by co-incubation with increasing concentrations of ISCU, and in cells incubated to steady state with 1 microM [3H]LY219703, ISCU (500 microM) rapidly displaced the photoaffinity analogue. Photoactivation of [3H]LY219703 by UV light (5-30 min) prevented efflux of radiolabeled drug during a 20-min wash in drug-free medium. Subsequent distribution studies showed that 89% of the radiolabel was associated with particulate components, and that approximately 20% of the radiolabel in the 320,000 g pellet could be extracted with acetone. Subcellular distribution showed approximately 6% associated with nuclei, 52% with mitochondria and 26% in the microsomal fraction. The effect of UV photoactivation on the distribution of [3H]LY219703 in soluble and particulate fractions was also examined in GC3/c1 cell preparations sonicated prior to being incubated with [3H]LY219703. A high proportion (83%) of radiolabel associated with the 100,000 g pellet, and distribution between soluble and particulate fractions was not altered by UV irradiation. Specific activities of protein in the 100,000 g supernatant and pellet were 0.186 and 0.537 nmol/mg, respectively. Putative binding species were analyzed by SDS-PAGE. Using SDS-PAGE, ten major binding proteins were identified in 320,000 g pellets from GC3/c1 cells: M(r) 110, 88, 76, 70, 64, 58, 48, 36, 26, and 24 kDa, and at least four of these (88, 70, 64, and 36 kDa) were also detected in mitochondria isolated from cells after photoactivation, or in mitochondrial preparations that were incubated with [3H]LY219703 and photoactivated after isolation from cells. Results suggested that under conditions of SDS-PAGE some dissociation of radiolabel from proteins also occurred. Binding of [3H]LY219703 to a model substrate, bovine serum albumin, and the effect of denaturing conditions used for sample preparation prior to SDS-PAGE, showed that relatively mild denaturing conditions (23 degrees, 2 hr) caused significant dissociation of radiolabel from BSA.(ABSTRACT TRUNCATED AT 400 WORDS)

Cross-resistance to antitumor diarylsulfonylureas and collateral sensitivity to mitochondrial toxins in a human cell line selected for resistance to the antitumor agent N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea.

Diarylsulfonylurea (DSU) antitumor agents represent a new class of oncolytic compounds with an unknown, potentially novel, mechanism of action. At high concentrations of several of these agents, cytotoxicity appears to be a consequence of uncoupling of mitochondria. However, the mechanism of action at pharmacologically achievable concentrations is unknown. To further study these agents a subline of human colon carcinoma, GC3/c1, was selected for resistance to N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) (Sulofenur). This clone (designated LYC5) was stably resistant for 2 years in the absence of selection pressure and was characterized for cross-resistance to other antitumor DSU and therapeutically used oncolytic agents. LYC5 was cross-resistant to six of seven DSU analogues examined when cells were exposed to drugs for 7 days. However, the degree of resistance was inversely related to the potency of the individual DSU against the parental GC3/c1 clone. Consequently, against LYC5 cells there was a relatively narrow range for concentrations inhibiting colony formation by 50% (4-fold), compared with that in GC3/c1 cells (12-fold range). With a single exception, each DSU examined caused uncoupling of oxidative phosphorylation in isolated mitochondria at 50 microM, and data suggest that cytotoxicity in LYC5 cells may be a consequence of mitochondrial impairment. In contrast, LYC5 cells were collaterally sensitive to the mitochondrial toxins rotenone, antimycin, and oligomycin, by 11.4-, 7.2-, and 36.9-fold respectively. LYC5 cells were also collaterally sensitive to vincristine (7.7-fold), Actinomycin D (5.9-fold), and rhodamine-123 (10.5-fold), agents associated with P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). LYC5 cells were slightly more sensitive to Melphalan and doxorubicin (2.8- and 2.3-fold, respectively) but not to cisplatin or dideazatetrahydrofolic acid. Collateral sensitivity to vincristine and Actinomycin D was consistent with decreased Pgp levels in LYC5 cells. Immunohistochemical staining and Western blotting with anti-Pgp antibodies indicated an 8-fold reduction in Pgp levels in LYC5 cells, relative to expression in parental GC3/c1 cells. Consequently, association of mitochondrial toxins with resistance in MDR KB8-5 cells was examined in the presence or absence of the MDR-reversing agent verapamil. KB8-5 cells had equal or greater sensitivity, compared with parental KB3-1 cells, to rotenone, antimycin, and oligomycin and also to each DSU analogue examined. In addition, verapamil tended to have a protective effect against these mitochondrial toxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Proliferation-dependent and -independent cytotoxicity by antitumor diarylsulfonylureas. Indication of multiple mechanisms of drug action.

The mechanism(s) by which antitumor diarylsulfonylureas (DSU) cause cytotoxicity has been examined in GC3/c1 human colon adenocarcinoma cells and a subline selected for resistance to N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU). Resistance was stable in the absence of selection pressure. This mutant (designated LYC5) was 5.5-fold resistant to ISCU compared to parental GC3/c1 cells in serum containing medium when cells were exposed for 7 days. In contrast, LYC5 cells were not resistant to a 4-hr exposure to ISCU. These data indicated two possible mechanisms of action, dependent on concentration and time of exposure to ISCU. Proliferation-dependent and -independent mechanisms of cytotoxicity were identified in wild-type and resistant clones. In serum-free medium containing growth factors, the IC50 for parental cells was 0.51 microM and for LYC5 7.0 microM (13.6-fold resistance), whereas without growth factors both lines were 8- to 9-fold resistant relative to conditions of cellular proliferation. Accumulation of ISCU was similar in quiescent and proliferating cells, and was reduced only slightly in resistant LYC5 cells. Analysis of DNA by agarose gel electrophoresis showed that in GC3/c1 cells nucleosomal ladders were formed only when proliferating cells were exposed to ISCU. No nucleosomal ladders were detected in quiescent cells during exposure to toxic concentrations of drug (IC90), or after removal of ISCU and addition of serum to stimulate growth. These data indicate several mechanisms by which diarylsulfonylurea antitumor agents may cause cell death. In serum-free medium at very high concentration (IC50 approximately 370 microM) for short periods of exposure (4 hr), cytotoxicity was proliferation independent, and GC3/c1 and LYC5 cells were equally sensitive. This mechanism may relate to the uncoupling activity of ISCU. However, at pharmacological relevant concentrations, the primary mechanism was proliferation dependent and led to formation of nucleosomal DNA ladders (IC50 approximately 0.5 microM). A possible additional mechanism occurred at higher concentration (IC50 approximately 7 microM) in quiescent cells, and was not associated with DNA degradation.

Influence of extracellular pH on the accumulation and cytotoxicity of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea in human cell lines.

The effect of extracellular pH (pH(e)) on the accumulation and cytotoxicity of the diarylsulfonylurea antitumor agent N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) has been examined. In a human colon adenocarcinoma cell line, GC3/C1, the initial rate of uptake of [3H]MPCU (2.4 microM) was increased by 4.5-fold as pH(e) was reduced from 7.4 to 6.5. Steady state levels of MPCU were inversely proportional to pH(e) and were 5-fold greater at pH 6.0 compared to 7.4. Similar results were obtained using Rh30 cells derived from an alveolar rhabdomyosarcoma. MPCU rapidly re-equilibrated after achieving steady state when pH(e) was altered, indicating that MPCU was not tightly bound within cells. In both cell lines, the uncoupling agent, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced (GC3/C1) or completely inhibited (Rh30) accumulation of MPCU at each pH(e) examined. Sodium azide had the same effect on the accumulation of MPCU as FCCP. The effects of FCCP and azide appeared to be due to collapse of the pH differential across the mitochondrial inner membrane rather than the gradient across the plasma membrane. As extracellular pH (pH(e)) decreased, intracellular pH(pH(i)) also decreased in GC3/C1 cells, such that the greatest pH differential (pH(i) - pH(e)) was 0.2 units at pH(e) 6.0. Neither FCCP nor azide significantly altered this pH gradient, indicating a minor role, if any, for the plasma membrane pH gradient in accumulation of MPCU in GC3/C1 cells. The effect of pH(e) (7.4 to 6.0) on cytotoxicity of MPCU was determined after exposure of cells for 4 hr to various concentrations of MPCU in the presence of 10% fetal bovine serum. Decreasing the pH(e) from 7.4 to 6.0 increased the potency of MPCU by 4.7- and 4.5-fold in Rh30 and GC3/C1 cells, respectively. In cells exposed to drug/pH(e) combinations that resulted in 50% reduction in colony forming potential, the steady state levels of [3H]MPCU were similar (range 8.8 +/- 0.9 to 10.56 +/- 0.6 nmol/10(6) cells). These results demonstrate that decrease of pH(e) significantly enhanced the uptake of MPCU accumulation into an FCCP/azide-sensitive compartment, and cytotoxicity of this agent. These data further support the hypothesis that sequestration of diarylsulfonylureas into the FCCP/azide-sensitive compartment (probably mitochondria) was associated with its cytotoxicity. The role of pH(e) in determining therapeutic selectivity of diarylsulfonylureas is discussed.

Conformational changes in myosin and heavy meromyosin from chicken gizzard associated with phosphorylation.

Heavy meromyosin (HMM) undergoes a conformational transition between a rapidly and a slowly sedimenting form, during which it sediments as a single peak in the ultracentrifuge with sedimentation coefficients between 7.5 and 9S. Changes in sedimentation velocity and ATPase activity produced by changes in ionic strength, phosphorylation of HMM or addition of MgATP are interpreted in terms of equilibria between the rapidly and slowly sedimenting forms, the observed values of activity and sedimentation velocity being determined by the fraction of HMM in each form. Phosphorylation of the 20 kDa light chain or raising the ionic strength decrease the sedimentation velocity, by decreasing the fraction of HMM in the rapidly sedimenting form, while addition of ATP increases sedimentation velocity upon forming a 9S HMM-ADP-Pi complex. Electron microscopic studies support this interpretation showing the presence of two distinct conformations of HMM--extended and flexed, which correspond to the 7.5S and 9S forms, respectively (Suzuki et al., 1985). In samples prepared at high ionic strengths, the heads extend away from the tail in a more or less random orientation, while at low ionic strength, the molecule is flexed at the head-tail junction assuming a more compact structure, that appears to account for its more rapid sedimentation rate. The degradation rates of the heavy chain and the 20 kDa light chain of HMM on digestion with papain indicate the presence of three forms of HMM differing in their susceptibility to papain. At 25 mM NaCl, HMM is rapidly digested in the absence of ATP, while addition of ATP decreases digestibility by a factor of ten, upon formation of a complex of HMM with the products of ATP hydrolysis. Above 0.4 M NaCl, HMM is degraded at an intermediate rate that is not affected by ATP. When the ionic strength is varied, the rate of disappearance of the heavy chain depends linearly on the sedimentation velocity in both the phosphorylated and dephosphorylated states, indicating that the rate of proteolysis is determined primarily by the fraction of HMM in the rapidly and slowly sedimenting forms. The same pattern is seen in the disappearance of the 20 kDa light chain of dephosphorylated HMM on cleavage at a site 4 kDa from the N-terminus, indicating that the cleavage of the light chain also depends on the fraction of HMM in the rapidly and slowly sedimenting forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Dual effect of filamin on actomyosin ATPase activity.

Filamin binds to F-actin and influences the myosin-actin interaction. At relatively low concentrations, filamin activates actomyosin Mg2+-ATPase, whereas higher concentrations of filamin exert an inhibitory effect. Activation of ATPase activity occurs under conditions where a loose meshwork of actin filaments is present and inhibition is associated with the appearance of closely apposed bundles of actin filaments. Maximum activation (about fourfold) of actomyosin ATPase activity by filamin occurs between 30 and 65 mM KCl, at pH 6.5, and at temperatures not less than 30 degrees C. ATPase activation requires higher concentrations of filamin in the presence than in the absence of tropomyosin. Filamin does not activate Mg2+-ATPase activity of acto-subfragment-1 and has only a slight effect on the Mg2+-ATPase of acto-heavy meromyosin, but it inhibits the activity of both these systems under conditions similar to those that inhibit actomyosin ATPase activity.

Potentiation of actomyosin ATPase activity by filamin.

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.

Dimerization of the polypeptide chains of skeletal muscle tropomyosin.

The composition of alpha and beta chains in tropomyosin dimers present in fetal and adult skeletal muscle of cow has been analysed by SDS-polyacrylamide gel electrophoresis after cross-linking of the chains by disulphide bridges. The results indicate that in vivo alpha beta heterodimers of tropomyosin are assembled preferentially and only the excess of particular chains forms homodimers, i.e., alpha alpha dimers in adult and beta beta ones in fetal muscle. The original dimers of tropomyosin were dissociated with urea in the presence of dithiothreitol. Subsequent reassembly of the tropomyosin dimers from the mixture of alpha and beta chains approaches the random model.