Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells.

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Critical targets of protein kinase C in differentiation of tumour cells.

The ultimate target of pharmacological research is to find new drugs for treating human diseases such as cancer. Agents causing differentiation and thus growth arrest should be particularly useful in this regard. A potential target for such anticancer therapy is the enzyme family protein kinase C (PKC), which is involved in the transduction of signals for cell proliferation, differentiation, and apoptosis. Our recent work showing the induction of differentiation in melanoma cells by an activator of one PKC isoform, PKCdelta, touches on several important areas of investigation, which will form the basis of this review: the role of individual isoforms of PKC, their downstream targets and their specific substrates, the mechanism of activation of specific genes involved in the differentiation process, and the molecular basis for the morphological changes associated with differentiation. The central role that PKC plays in these processes points to the need for a greater understanding of the signalling pathways utilized by individual isoforms of this family of enzymes.

Bistratene A induces a microtubule-dependent block in cytokinesis and altered stathmin expression in HL60 cells.

Bistratene A is a cyclic polyether which affects cell cycle progression and can induce phosphorylation of cellular proteins. Treatment of HL60 cells with 100 ng/ml bistratene A was found to inhibit cytokinesis but had no effect on DNA synthesis and nuclear division. Consequently, bistratene A-treated cells became polyploid and multinucleate. In association with the development of this phenotype, the cytoplasmic protein stathmin was biphasically phosphorylated and levels of expression were doubled. Immunostaining of binucleate cells (bistratene A for 24 h) revealed increased alpha-tubulin localization where the cleavage furrow might be expected to form, i.e., along the equatorial plane. Treatment of these binucleate cells with the microtubule depolymerizing agent nocadazole promoted cleavage furrow formation and partially ameliorated the bistratene A-induced block in cell division. These findings implicate the polymerization status of microtubules and stathmin function in the regulation of cytokinesis.

Calpain activation is upstream of caspases in radiation-induced apoptosis.

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling.

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.

Endocytosis by digest cells of the cattle tick Boophilus microplus: regulation by protein kinase C.

Endocytosis of fluorescently-labeled bovine serum albumin by digest cells of the gut of the cattle tick Boophilus microplus is inhibited by approx 60% in the presence of the tumour promoter 12-O-tetradecanoylphorbol 13-acetate. The results are consistent with a role for protein kinase C in regulating the uptake of blood meal by digest cells. Protein kinase C activity has been measured in the digest cell and the amount of enzyme has also been determined using a phorbol ester binding assay. The presence of a small number of specific protein kinase C substrates in the plasma membrane of the digest cell has been demonstrated. Preliminary experiments indicate that one of these substrates, a protein of approximately 30 kDa, is an integral membrane protein, part of which is exposed on the extracellular surface of the digest cell.

Accumulation of HL-60 leukemia cells in G2/M and inhibition of cytokinesis caused by two marine compounds, bistratene A and cycloxazoline.

The effects on the cell cycle of two biologically active compounds, bistratene A and cycloxazoline, from the marine ascidian Lissoclinum bistratum were studied in HL-60 human leukemia cells using flow cytometry. Both compounds were shown to cause an apparent accumulation of cells in the G2/M phase. This effect was shown to be both time- and dose-dependent. At the longer time points (30 and 48 h after addition of the compounds) polyploidy was apparent. The fate of cells labeled in the S phase with 5-bromo-2'-deoxyuridine (BrdUrd) was analysed using a bivariate BrdUrd/PI (propidium iodide) technique. Bistratene A and cycloxazoline treatment prevented the majority of BrdUrd-labeled cells from progressing through to the G1 phase. Approximately 50% of the cells were delayed at G2/M, and a significant proportion of cells appeared to be polyploid. Light and electron microscopy revealed the presence of multinucleated cells accounting for the apparent polyploidy. The progression of cells out of the G1 phase was also examined by synchronising cells with mimosine and releasing them from mimosine block in the presence of bistratene A. There was no evidence of a block at the G1/S phase transition or through the S phase since DNA synthesis was not inhibited. The mechanism by which these compounds interfere with cytokinesis is presently unknown but, in the case of bistratene A, may be linked to altered phosphorylation of cellular proteins involved in cell-cycle control.

Toxins from ascidians.

A wide range of toxins with interesting pharmacological properties have been isolated from ascidians. These include cyclic peptides, one of which, didemnin B, is undergoing clinical trials for cancer chemotherapy and is also a powerful immunosuppressive agent. Other classes of toxins from these organisms are alkaloids, macrolides, polyethers, prenylated hydroquinones and others of diverse chemical structure. The majority of these compounds are cytotoxic and in some cases the mechanisms of action have been investigated. The toxins are most likely used for chemical defense but the question of how the ascidians themselves escape the toxic actions is also not resolved.

Bistratene A: a novel compound causing changes in protein phosphorylation patterns in human leukemia cells.

Bistratene A, a polyether toxin isolated from the colonial ascidian Lissoclinum bistratum, causes incomplete differentiation of human leukemia (HL-60) cells apparently through a mechanism not involving protein kinase C. In view of the importance of phosphorylation/dephosphorylation in cellular growth and differentiation we have investigated protein phosphorylation in these cells following exposure to bistratene A, using two-dimensional polyacrylamide gel electrophoresis. Marked increases in the phosphorylation of a protein of 20 kDa, pl 6.7, and a basic protein of 25 kDa were observed after incubation with bistratene A. A comparison was made with cells treated with 12-O-tetradecanoylphorbol 13-acetate and bryostatin 5. While changes in phosphorylation patterns were observed with these two compounds, the 20 kDa and 25 kDa proteins did not undergo phosphorylation changes. The 20 kDa protein was induced rapidly by very low concentrations of bistratene A reaching near maximal levels with 10 nM at 15 min exposure. This protein was found to be localised to the cytoplasm. Phosphoaminoacid analysis demonstrated that the majority of 32P was present in serine and tyrosine residues. The increased phosphorylation of the 20 kDa protein appeared to be due to hyperphosphorylation of existing protein although there was some increase in the amount of the protein. These results suggest that bistratene A will be a useful tool with which to investigate cellular differentiation mechanisms.

Structure-activity relationships of the lissoclinamides: cytotoxic cyclic peptides from the ascidian Lissoclinum patella.

Two new lissoclinamides (lissoclinamides 7 and 8) have been isolated from the aplousobranch ascidian Lissoclinum patella. These lissoclinamides are cyclic heptapeptides with the same structural features as lissoclinamides 4 and 5 reported earlier, containing an oxazoline ring, one proline, one valine, two phenylalanine residues, and thiazole and/or thiazoline rings. All four peptides have the same sequence of amino acids around the ring and differ from one another only in their stereochemistry or the number of thiazole and thiazoline rings. The cytotoxicities of the compounds were tested with human fibroblast and bladder carcinoma cell lines and normal lymphocytes. Slight changes in structure resulted in marked differences in the cytotoxicities of these compounds. The most potent is lissoclinamide 7, containing two thiazoline rings, which rivals didemnin B in cytotoxicity in vitro.

New cyclic peptides with cytotoxic activity from the ascidian Lissoclinum patella.

The isolation and structures of a new patellamide (patellamide D) and two new lissoclinamides (lissoclinamides 4 and 5) from the aplousobranch ascidian Lissoclinum patella are described. Structures were determined largely by using two-dimensional NMR techniques and mass spectrometry. These peptides and other members of the patellamide and lissoclinamide families that have been reported previously are found within the obligate algal symbiont of the genus Prochloron. The cytotoxicities of the compounds toward fibroblast and tumor cell lines are reported. One of these compounds, lissoclinamide 4, is markedly more toxic than other members of the family. Structure-activity relationships are discussed.

Novel cytotoxic compounds from the ascidian Lissoclinum bistratum.

The isolation and structures of two new cyclic hexapeptides and two new macrocyclic ethers from the aplousobranch ascidian Lissoclinum bistratum are described. Their structures were determined by two-dimensional NMR techniques. The hexapeptides, named bistratamide A and bistratamide B, differ only by the presence or absence of one double bond. They were tested for cytotoxicity toward human fibroblast and tumor cell lines and displayed similar toxicities to the octapeptides called patellamides from Lissoclinum patella. The peptides are found within the obligate algal symbiont Prochloron but clearly differ from peptides isolated from the same Prochloron of L. patella. The macrocyclic ethers isolated from L. bistratum are exceedingly potent in cytotoxicity. They have been named bistratenes A and B, and structures for these compounds are proposed.

A monoclonal antibody to a parasympathetic neurotrophic factor causes immunoparasympathectomy in mice.

A monoclonal antibody capable of blocking the biological activity of the ciliary neurotrophic factor purified from bovine cardiac muscle has been produced. This antibody, when administered perinatally to mice, causes a failure of the normal development of the parasympathetic innervation of the iris as determined by assay for the activity of the cholinergic marker enzyme choline acetyltransferase. The same treatment has no effect on the adrenergic neuronal marker, tyrosine hydroxylase. This immunoparasympathectomy suggests that the ciliary neurotrophic factor has an essential role in regulating the development of the mammalian parasympathetic nervous system.

Purification of a ciliary neurotrophic factor from bovine heart.

A neurotrophic factor that promotes the survival of cholinergic parasympathetic ciliary neurons has been purified approximately 20,000-fold from bovine cardiac tissue under nondenaturing conditions using heparin-affinity chromatography. Up to 22 micrograms of purified factor having a specific activity of 4 X 10(5) trophic units/mg can be obtained from 250 g of heart muscle. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified material show a broad band that is sometimes resolvable into a closely spaced pair of bands of 22 and 23 kilodaltons. Partially purified factor can be resolved into two peaks of activity (pI 5.6 and 5.0) by high-resolution anion-exchange chromatography and chromatofocusing, although these procedures have not proved useful as purification methods because of the large losses of activity incurred. It is likely that these two peaks represent the two bands seen on SDS-polyacrylamide gels. The bovine cardiac factor(s) differs from similar factors purified from chick optic tissues and pig brain in that it is irreversibly denatured by SDS.

Long-term retention of Fast Blue in sympathetic neurones after axotomy and regeneration--demonstration of incorrect reconnections.

Using the fluorescent dyes Fast Blue and Diamidino Yellow to trace neuronal connections, the regeneration of the superior cervical ganglionic neurones after axotomy has been examined. Fast Blue has the property of remaining within the neuronal perikaryon for many months after its retrograde axonal transport, even after transection of the axon. Thus, Fast Blue can be used to label neurones as to their original target and Diamidino Yellow subsequently used to demonstrate the specificity of reconnection. The results suggest that apparent return of appropriate function occurs in the presence of a large component of inappropriate reconnections.

Sympathetic neuronal survival factors change after denervation.

The increase in sympathetic neuronal survival factors in the hearts of rats chemically denervated with 6-hydroxydopamine has been examined after biochemical fractionation techniques. Affinity chromatography and gel filtration of extracts of control hearts have demonstrated the existence of two major components, neither of which is nerve growth factor (NGF). The concentrations of both of these components and of NGF are increased significantly after denervation.

An analysis of peripheral neuronal survival factors present in muscle.

Extracts of bovine heart, rat heart, and rat skeletal muscle were chromatographically separated and the fractions tested for their ability to maintain the survival of dissociated sympathetic, parasympathetic, and sensory neurones. Bovine heart contained at least five, rat heart at least six, and rat skeletal muscle at least four active components differing in their physicochemical properties and their target selectivity. Bovine and rat heart appeared to contain at least two components in common. The differences in active components found in the three tissues are consistent with a complex and perhaps tissue-specific system regulating neuronal survival.

Effects of cardiac denervation on levels of neuronal survival factors for cultured autonomic neurones.

The content of survival factors for sympathetic, sensory and parasympathetic neurones in the heart was examined after denervation with either 6-hydroxydopamine or vagal transection. There was a correlation between the type of nerve sectioned in vivo and the type of neurone whose survival in vitro was enhanced by the presence of denervated target tissue.