Impaired fracture healing in macrophage migration inhibitory factor-deficient mice.

UNLABELLED: This study investigated the role of macrophage migration inhibitory factor (MIF) in fracture repair using MIF gene-deficient mice (MIF KO). Fracture healing was delayed in MIF KO, and this was mainly due to the delay in the mineralization of osteoid within the fracture callus. INTRODUCTION: We previously reported that the expression of macrophage migration inhibitory factor (MIF) was up-regulated during the fracture healing process in rats. However, its role in the pathophysiology of this process remained unclear. The aim of the present study was to clarify the role of MIF in the fracture healing process using MIF gene-deficient mice (MIF KO). METHODS: Bone repair in wild-type mice (WT) and MIF KO (n = 70, respectively) was investigated using a tibia fracture model. Radiographic, biomechanical, histological, bone histomorphometric, and molecular analyses were performed. RESULTS: Post-fracture biomechanical testing showed that maximum load and stiffness were significantly lower in MIF KO than in WT on day 42. However, similar levels were observed between the two groups on day 84. Bone histomorphometric analysis revealed significantly higher osteoid volume, a lower mineral apposition rate, and smaller numbers of osteoclasts in the MIF KO callus compared to the WT callus. The messenger ribonucleic acid expressions of matrix metalloproteinase (MMP)-2, membranous type 1-MMP, cathepsin K, and tissue nonspecific alkaline phosphatase were found to be significantly suppressed in the MIF KO callus. CONCLUSION: The results of the present study suggest that delayed fracture healing in MIF KO was mainly attributable to a delay in osteoid mineralization.

Precise absolute Seebeck coefficient measurement and uncertainty analysis using high-Tc superconductors as a reference.

The intrinsic properties of superconductors enable the direct determination of the absolute Seebeck coefficient at low temperature due to the disappearance of the Seebeck effect to obey the Meissner effect. We report a precision absolute Seebeck coefficient measurement for the fine Pt sample determined using the high-Tc YBa2Cu3O7-x (YBCO) superconductor as a reference and an analysis of the measurement uncertainty. To make a precision measurement and aid in the verification of the uncertainty components, we developed a cryostat system that enables temperature control in a stable manner. The expected performance of the reference superconductor yielded a zero value well below Tc, which was validated by a superconductor-superconductor thermocouple experiment. Uncertainty analysis shows that the main limiting factor for this measurement is the accuracy of the temperature difference measurement using the resistance temperature sensors, along with its analog noise. We obtained values of S = 5.6 ± 0.2 µV/K with a relative expanded uncertainty of 3% at 80 K and precisely compared the Pt value with that determined by the high-Tc Bi2Sr2Ca2Cu3O8+δ (Bi-2223) superconductor, which has a higher Tc. We found that there was no difference between the Seebeck coefficient values obtained from the YBCO and Bi-2223 references up to its Tc within the expanded measurement uncertainties of 0.3 µV/K (2σ). These results provide accurate validation that the high-Tc superconductor is a useful reference up to the liquid nitrogen temperature.

A new tumor-promoting agent, dihydroteleocidin B, markedly enhances chemically induced malignant cell transformation.

Teleocidin, which was isolated from mycelia of Streptomyces, is a potent tumor promoter in mouse skin. The catalytically hydrogenated compound dihydroteleocidin B markedly enhanced malignant cell transformation induced by 3-methylcholanthrene or ultraviolet radiation. Dihydroteleocidin B was at least 100 times more effective in enhancing transformation than 12-O-tetradecanoyl phorbol-13-acetate, the strongest promoter known until now, whereas both promoters showed equal capacities to induce early membrane effects and DNA synthesis.

Bromine residue at hydrophilic region influences biological activity of aplysiatoxin, a tumor promoter.

Aplysiatoxin and debromoaplysiatoxin, which are isolated from the seaweed, Lyngbya gracilis, differ in their chemical structure only by the presence or absence of a bromine residue in the hydrophilic region. The function and the structure-activity relation of the hydrophilic region are not known. Aplysiatoxin increased malignant transformation, stimulated DNA synthesis, and inhibited the binding of phorbol-12,13-dibutyrate and epidermal growth factor to cell receptors. Debromoaplysiatoxin inhibited the binding of these two substances as strongly as aplysiatoxin but did not increase malignant transformation or stimulate DNA synthesis. These results indicate that a slight change in the chemical structure of the hydrophilic region of aplysiatoxin affects its abilities to increase cell transformation and stimulate DNA synthesis and that the abilities of the tumor promoters to inhibit the binding of phorbol-12,13-dibutyrate and epidermal growth factor are dissociable from their abilities to increase cell transformation and stimulate DNA synthesis under some circumstances.

Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication.

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade). When an A*-N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*-N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3' sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3' and 5'-TA*A-3' sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pair may be an important factor for the mutation spectra of 2-OH-Ade.

Effects of tumor promoters on the frequency of metallothionein I gene amplification in cells exposed to cadmium.

Three potent tumor promoters of different classes, 12-O-tetradecanoylphorbol-13-acetate, dihydroteleocidin B, and aplysiatoxin, and two moderate tumor promoters, mezerein and debromoaplysiatoxin, enhanced the frequency of appearance of cadmium-resistant Chinese hamster lung cells when the cells were exposed to cytotoxic levels of CdCl2. With these compounds, the activity to induce cadmium-resistant cells correlated well with the potency of tumor-promoting activity. Cadmium resistance, which persisted after removal of the tumor promoters, was associated with the overproduction of metallothionein I messenger RNA. The amplified metallothionein I genes were shown by Southern blotting experiments. The relevance of the gene amplification caused by tumor promoters is discussed in relation to cancer development and progression.

Induced secretion of a Mr 46,000 protein by cultured human hepatoma cells treated with tumor promoters.

Human hepatoma cells, HuH-6 Cl-5, were treated with 12-O-tetradecanoylphorbol-13-acetate at concentrations of 1 ng/ml to 10 micrograms/ml for 6 h in the presence of [35S]methionine. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled proteins secreted into the medium showed that this treatment induced marked secretion of a polypeptide with a molecular weight of about 46,000 (p46). When the labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis, p46 was composed of three isoproteins which had different isoelectric points. The two new classes of tumor promoters, teleocidin and aplysiatoxin, also induced secretion of this protein, although the amount of p46 induced by debromoaplysiatoxin was less than that induced by 12-O-tetradecanoylphorbol-13-acetate, teleocidin, and aplysiatoxin, judging from densitometric scanning of bands on a fluorogram. The nonpromoting phorbol esters, such as 4 beta-phorbol and phorbol-13-monoacetate, did not induce p46. The induction of p46 secretion involved de novo RNA synthesis, since actinomycin D (1 microgram/ml) completely and selectively blocked the incorporation of [35S]-methionine into the protein. "Pulse-chase" experiments indicated that p46 was not a degradation product induced by these potent tumor promoters. In an attempt to identify p46, the total proteins released were treated with two kinds of rabbit anti-human whole serum antisera, but although some proteins were precipitated, p46 was not.

Heterologous regulation of the epidermal growth factor receptor by palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter.

Previous results have established that 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters can alter the properties of the epidermal growth factor (EGF) receptor through activation of protein kinase C. In order to determine whether other, non-TPA-type tumor promoters might similarly influence growth-mediating receptors, we investigated the effect of palytoxin on EGF binding in Swiss 3T3 fibroblasts and human epidermal carcinoma (A431) cells. In both cell types, pretreatment with a low dose of palytoxin (1-11 pM) at 37 degrees C causes a decrease in EGF binding. In Swiss 3T3 cells the inhibitory effect is temperature dependent and does not occur at 4 degrees C, indicating that palytoxin is not directly competing with EGF for binding. As assessed by effects on DNA synthesis, palytoxin is not toxic at these concentrations and does not appear to be mitogenic for these cells. Although palytoxin, like phorbol esters, alters EGF binding, its action in Swiss 3T3 cells differs from that of TPA-type tumor promoters in at least 4 respects: (a) the kinetics and dose dependence differ significantly from that of phorbol dibutyrate; (b) the effect is not readily reversible; (c) there is loss of low-affinity as well as high-affinity binding sites; (d) the effect is independent of cellular protein kinase C levels. These results indicate that palytoxin is capable of heterologous regulation of the EGF receptor through a novel mechanism and suggest that certain non-TPA-type tumor promoters as well as TPA-type tumor promoters may act in part through modulation of growth regulatory pathways.

Distinct inhibitory effects of dihydroteleocidin B and the phorbol ester tumor promoters on the adipocyte differentiation of 3T3-L1 cells.

An indole alkaloid tumor promoter, dihydroteleocidin B, was able to modulate a membrane property of 3T3-L1 preadipocytes, showing an almost complete reduction of epidermal growth factor binding capacity. This receptor modulating potency of dihydroteleocidin B, was 10 times that of a phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Dihydroteleocidin B, however, had little effect on the epidermal growth factor receptors of the adipocyte stage of 3T3-L1. Adipocyte differentiation was induced by treating growth-arrested 3T3-L1 cells with dexamethasone and 1-methyl-3-isobutylxanthine for 48 hr. These inducers initiated DNA synthesis, led to one full cycle of cell division, and triggered the adipocyte differentiation program. Dihydroteleocidin B almost completely inhibited this differentiation at concentrations of 1 to 10 ng/ml (10(-9) to 10(-8) M). The inhibition was observed regardless of when the tumor promoter was added: before, during, or after the addition of inducers. Similar inhibition was also observed by TPA, but with over 90% less efficiency than that of dihydroteleocidin B. TPA was most effective when it was added during the inducer treatment. Both dihydroteleocidin B and TPA stimulated DNA synthesis to the same level during the initial 22 hr. The DNA synthesis stimulated by dihydroteleocidin B resulted in extraordinary enhancement of cell proliferation, whereas TPA-treated 3T3-L1 cells did not divide. These findings suggest that dihydroteleocidin B and TPA have distinct potencies in interfering with the mechanisms of adipocyte differentiation and that presumably they are different in action of tumorigenesis.

Mouse 3T3-L1 cell variants unable to respond to mitogenic stimulation of dihydroteleocidin B: genetic evidence for the synergism of tumor promoters with growth factors.

Dihydroteleocidin B, an indole alkaloid tumor promoter, stimulates confluent, quiescent mouse 3T3-L1 fibroblasts to initiate DNA synthesis and undergo cell division. Using a mitotic shakeoff technique, we have isolated 12 clones of genetic variants which are unable to respond to the mitogenic stimulation of dihydroteleocidin B from a total of 12 million cells. Biochemical characterization of these nonresponsive variants to dihydroteleocidin B revealed that there is no change in the ability to bind [3H]phorbol dibutyrate, the activity of protein kinase C, and the turnover of phosphatidylinositol. The evidence indicates that nonresponsiveness to dihydroteleocidin B is caused by several different lesions, including defects in receptors for insulin or epidermal growth factor and in the postreceptor mechanisms. The evidence also suggests that mitogenic signal transfer via the epidermal growth factor receptor system appears to share a common step with dihydroteleocidin B whereas the signal transfer for insulin seems separate from these. These results suggest that phosphatidylinositol turnover followed by protein kinase C activation alone is not sufficient for mitogenic stimulation and that the coordination of the protein kinase C system with the receptor systems for growth factors may be necessary for "full" mitogenic response.

Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells treated with tumor-promoting indole alkaloids or polyacetates.

The synthesis of a unique protein with a molecular weight of 32,000 (p32) in BALB/c 3T3 cells has been shown previously to increase after treatment with potent tumor-promoting phorbol esters (Hiwasa et al., Proc. Natl. Acad. Sci. U. S. A., 79: 1800, 1982). In the present study, two new classes of tumor promoters which are structurally different from phorbol esters were investigated for their potencies to enhance p32 synthesis. Teleocidin, dihydroteleocidin B, and lyngbyatoxin A, which are indole alkaloid tumor promoters, enhanced p32 synthesis to the same extent that 12-O-tetradecanoylphorbol-13-acetate did. However, no increase was observed by treatment with the biologically inactive hydrolysate of teleocidin. Polyacetate tumor promoters such as aplysiatoxin and debromoaplysiatoxin also stimulated p32 synthesis, but their effective concentrations were higher than those of 12-O-tetradecanoylphorbol-13-acetate. When 3T3 cells were treated with a combination of two of the three tumor promoters, TPA, teleocidin, and aplysiatoxin, no synergistic effect of p32 synthesis was observed. This implies that these tumor promoters enhance the synthesis of p32 through the same mechanism.

Regulation of the epidermal growth factor receptor by growth-modulating agents: effects of staurosporine, a protein kinase inhibitor.

Staurosporine is a potent microbial inhibitor of a number of protein kinases, including protein kinase C, cyclic AMP-dependent kinase, and the tyrosine kinase pp60src. We have used staurosporine to investigate the role of phosphorylation in the regulation of the epidermal growth factor (EGF) receptor in both human epidermal carcinoma A431 cells and mouse Swiss 3T3 fibroblasts. We report here that staurosporine treatment causes enhancement in high affinity EGF binding and a decrease in the phosphorylation state of the unstimulated receptor at a number of residues, including threonine 669. Staurosporine also antagonizes the inhibition of high affinity EGF binding and the increase in phosphorylation state of the unstimulated EGF receptor by phorbol esters and the calcium ionophore A23187. Staurosporine is an effective inhibitor of the EGF-stimulated receptor tyrosine kinase in vitro and thus does not enhance EGF stimulation of EGF receptor autophosphorylation in vivo. These results suggest that phosphorylation plays a major role in the regulation of the high affinity binding state of the EGF receptor in both unstimulated and mitogenically activated cells.

Inhibition of methylnitrosourea-induced large bowel cancer development in rats by sarcophytol A, a product from a marine soft coral Sarcophyton glaucum.

An antitumorigenic effect of sarcophytol A (SaA), a simple monohydroxycembratetraene isolated from a marine soft coral Sarcophyton glaucum, was investigated in rat colon carcinogenesis. Three groups (26 rats each) of female CD-Fischer rats given an intrarectal dose of 2 mg of N-methyl-N-nitrosourea 3 times weekly for Wk 1 to 3 were fed standard laboratory chow in the control group or the chow containing 0.01% SaA from Wk 1 or from Wk 4 in experimental groups. The body weight gain and the food intake were not different among all 3 groups, and SaA intake was similar in both experimental groups at a dosage of 6.18 and 6.14 mg/kg of body weight/day at Wk 5 and 3.87 and 3.90 mg/kg of body weight/day at Wk 25. At autopsy at Wk 26, the incidence of large bowel tumors was found to be significantly lower and the mean number of tumors per tumor-bearing rat to be insignificantly smaller in experimental groups than in the control group: 50% and 58% versus 85%, 1.8 and 1.8 versus 2.0. The tumors in both experimental groups were generally smaller. All the tumors except two signet ring cell carcinomas were well-differentiated adenocarcinomas. Induction of ornithine decarboxylase activity, a marker of tumor promotion, in the large bowel mucosa of rats which were fed the SaA chow for 1 wk, then received an intrarectal dose of 12, 6, or 1.2 mumol of deoxycholate, a tumor promoter in large bowel carcinogenesis, and were killed 4 h later was significantly lower than in control rats. Thus, it was concluded that SaA inhibited the development of large bowel cancer, probably through an antipromoting mechanism.

Synergistic effects of (--)-epigallocatechin gallate with (--)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9.

The study on incorporation of [3H](-)-epigallocatechin gallate (EGCG) into human lung cancer cell line PC-9 indicated that the [3H]EGCG incorporation was significantly enhanced by (-)-epicatechin, an inert tea polyphenol without a galloyl moiety. (-)-Epicatechin enhanced apoptosis, growth inhibition of PC-9 cells, and inhibition of tumor necrosis factor-alpha release from BALB/c-3T3 cells by EGCG and other tea polyphenols with a galloyl moiety in a dose-dependent manner. Moreover, the effects of EGCG on induction of apoptosis were also synergistically enhanced by other cancer-preventive agents, such as sulindac and tamoxifen. This paper reports significant evidence that whole green tea is a more reasonable mixture of tea polyphenols for cancer prevention in humans than EGCG alone and that it is even more effective when it is used in combination with other cancer preventives.

Hyperphosphorylation of retinoblastoma protein and p53 by okadaic acid, a tumor promoter.

A potent tumor promoter, okadaic acid, induced hyperphosphorylation of tumor suppressor proteins, retinoblastoma protein and p53, by in vitro incubation with nuclei isolated from rat regenerating liver as well as by incubation with primary human fibroblasts. Most of the retinoblastoma protein migrated to a hyperphosphorylated position in electrophoresis. The phosphorylation of p53 was increased at a rate 8 times that in non-treated primary human fibroblasts. Hyperphosphorylation of tumor suppressor proteins, mediated through inhibition of protein phosphatases 1 and 2A, is involved in tumor promotion by okadaic acid. The significance of hyperphosphorylation of the retinoblastoma protein and p53 is discussed in relation to the regulation of the cell cycle.

Absence of synergistic effects on tumor promotion in CD-1 mouse skin by simultaneous applications of two different types of tumor promoters, okadaic acid and teleocidin.

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, and teleocidin, an activator of protein kinase C, are both potent tumor promoters on mouse skin. The effects of simultaneous treatment of the two different types of tumor promoters on tumor promotion as well as on their biochemical activities were studied. Three independent experiments with different doses of tumor promoters revealed that simultaneous repeated applications of okadaic acid and teleocidin did not induce any synergistic or additive effects on tumor promotion in mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). In Experiment 1, the group treated with a single application of DMBA, followed by repeated applications of 1.0 micrograms (1.2 nmol) okadaic acid and 2.5 micrograms (5.7 nmol) teleocidin, resulted in 64.3% tumor-bearing mice at week 20. But the groups treated with DMBA plus okadaic acid or DMBA plus teleocidin gave 73.3% and 71.4%, respectively. The biochemical activities were studied by means of induction of ornithine decarboxylase in mouse skin and protein phosphorylation in the cells. Simultaneous application of okadaic acid at three different doses with teleocidin did not induce ornithine decarboxylase activity synergistically or additively. Phosphorylation of proteins, cytokeratins, or heat shock protein 27 was not synergistically increased in human keratinocytes treated with okadaic acid and teleocidin, although the cotreatment in a cell-free system synergistically increased protein phosphorylation. Thus, the absence of synergistic effects on tumor promotion in mouse skin was also confirmed in two systems, induction of ornithine decarboxylase in mouse skin and protein phosphorylation in human keratinocytes. The effect of cotreatment of okadaic acid and teleocidin is discussed at the molecular level.

Heterogeneous nuclear ribonucleoprotein B1 as a new marker of early detection for human lung cancers.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an RNA binding protein that is required for maturation of mRNA precursor. Tockman et al. previously reported that hnRNP A2/B1 with a M(r) of 31,000 is overexpressed from the early clinical stage of human lung cancer (M. S. Tockman et al., J. Clin. Oncol., 6: 1685-1693, 1988). However, when hnRNP A2/B1 mRNA and hnRNP B1 mRNA were separately studied, we found unique evidence that hnRNP B1 mRNA, which is a splicing variant of hnRNP A2 mRNA, was more significantly elevated in lung cancer tissues than hnRNP A2/B1 mRNA. Our hnRNP B1-specific polyclonal antibody specifically recognized hnRNP B1 protein as a M(r) 37,000 nuclear protein by Western blotting but did not recognize hnRNP A2 protein. Immunohistochemical staining with the hnRNP B1 antibody revealed that hnRNP B1 protein was specifically stained in the nuclei of human cancer cells, and in squamous cell carcinomas in particular, but not in those of normal adjacent lung epithelial cells. We think that hnRNP B1 protein of M(r) 37,000, not hnRNP A2, is well qualified as a biomarker for the detection of human lung cancer.

A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol.

A new type of phorbol ester, which has a macrocyclic dicarboxylic acid diester structure, was isolated from the seed oil of Jatropha curcas L. (Euphorbiaceae). Based on the results of spectroscopic analyses of the compound and its chemical degradation products, its structure is proposed to be an intramolecular 13,16-diester of 12-deoxy-16-hydroxyphorbol, 12-deoxy-16-hydroxyphorbol-4'-[12',14'-butadienyl]-6'-[16',18',20' - nonatrienyl]-bicyclo[3.1.0]hexane-(13-O)-2'-[carboxylate]-(16-O)-3 '- [8'-butenoic-10']ate (DHPB). DHPB showed slightly weaker biological and biochemical activities than 12-O-tetradecanoylphorbol-13-acetate (TPA). DHPB induced ornithine decarboxylase in mouse skin (2.8 nmol CO2/30 min/mg protein/34 nmol application), inhibited the specific binding of [3H]-12-O-tetradecanoylphorbol-13-acetate to phorbol ester receptors (50% effective dose, 17.0 nM), and activated protein kinase C in vitro (50% effective dose, 36.0 nM). Also, a weak tumor-promoting activity of DHPB was found in a two-stage carcinogenesis experiment on mouse skin. One week after initiation of mice with 100 micrograms of 7,12-dimethyl-benz(a)anthracene, topical application, twice a week, of 2 micrograms of DHPB until week 17, followed by application of 5 microgram of DHPB until week 30 at the same rate, resulted in 46.7% incidence of tumors by week 30. The groups treated with 7,12-dimethylbenz(a)anthracene alone or DHPB alone did not produce significant numbers of tumors. These results indicate that the new phorbol ester, DHPB, is a tumor promoter with weaker activity than 12-O-tetradecanoylphorbol-13-acetate.

Structure-activity studies of the nonphorbol tumor promoter palytoxin in Swiss 3T3 cells.

Derivatives of palytoxin have been prepared which are modified on either the hydroxyl terminus or the amino terminus of the molecule. Previously we have shown that palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter, can inhibit epidermal growth factor binding in Swiss 3T3 cells through a pathway which is sodium dependent but not calcium or protein kinase C dependent. We used the epidermal growth factor receptor system to determine whether the specific chemical modifications of palytoxin present in these derivatives alter the cellular mechanism of action of the toxin. The dose response and ion dependence of palytoxin, the hydroxyl terminus derivative palytoxin-COOH, and the amino terminus derivatives N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were compared with respect to inhibition of epidermal growth factor binding. The potency of palytoxin-COOH was similar to that of palytoxin. By contrast, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were approximately 1/100 as potent as palytoxin in this assay. All three derivatives were at least 100-fold less toxic than palytoxin. Like palytoxin, the activities of palytoxin-COOH, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were dependent upon the presence of extracellular sodium. However, there was a significant difference in the dependence of the derivatives on extracellular calcium. Our results suggest that the hydroxyl terminus is important for determining the calcium dependence of the molecule and the amino terminus is important for determining the biological potency of palytoxin. We conclude that modification of the hydroxyl terminus region is an effective means of reducing the toxicity of palytoxin while retaining the biological effects.

Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin.

Staurosporine, which is a potent inhibitor of protein kinases, such as protein kinase C, inhibited both inductions of adhesion of human promyelocytic leukemia cells (50% effective dose = 9.0 nM) and Epstein-Barr virus early antigen in Raji cells (50% effective dose = 3.4 nM) by teleocidin. However, staurosporine induced irritation on mouse ear and histidine decarboxylase activity in mouse skin. It did not induce ornithine decarboxylase activity in mouse epidermis. The two-stage carcinogenesis experiments of staurosporine were carried out at two different doses. Experiment 1 revealed that the group treatment with a single application of 100 micrograms of 7,12-dimethylbenz(a)anthracene, followed by repeated applications of 50 micrograms of staurosporine, resulted in 85.7% of tumor-bearing mice at Wk 30, whereas group treatment with staurosporine alone or 7,12-dimethylbenz(a)anthracene alone gave 6.7% and 0%, respectively. Experiment 2 showed that group treatment with 7,12-dimethylbenz(a)anthracene followed by applications of 10 micrograms of staurosporine resulted in 33% of tumor-bearing mice at Wk 30. In addition, staurosporine treatment reduced the percentages of tumor-bearing mice treated with teleocidin from 100% to 67% in Wk 15. These results demonstrated that staurosporine is a weak tumor promoter of mouse skin compared with teleocidin, but staurosporine has some potency to inhibit tumor promotion by teleocidin.