Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells.

Tumour-derived factors suppress differentiation and function of in vitro generated DC. Here, we investigate the effect of two melanoma clones differing in their invasive and metastatic properties on the generation and/or functional maturation of human epidermal LC. LC were generated from CD34(+) cord blood progenitors under GM-CSF/TNF-alpha/TGF-beta 1. CD34(+) cells were co-cultured with or without melanoma cells using Transwell dishes. After 11 days of co-culture, CD34(+)-derived cells display a non-adherent undifferentiated morphology, a high level of monocytic CD14 marker, a down-regulated expression of LC markers (CD1a, E-cadherin) and DC markers (CD40, CD80, CD54, CD58, CD83, CD86, HLA-DR, HLA-class I). These cells were less potent than control LC in inducing allogeneic T cell proliferation. The generation of the CD14(+) population was correlated with a decrease in the CD1a(+) population, without any statistical differences between the two clones. Melanoma cells diverted the differentiation of CD34(+) cells towards a dominant CD14(+) population only if the progenitors were in an early growth phase. IL-10, TGF-beta 1 and VEGF were not responsible for these effects, as assessed by using blocking antibodies. By contrast, co-culture of fresh epidermal LC with melanoma cells did not affect their phenotype and function. Our data demonstrate that melanoma cells inhibit the earliest steps of LC differentiation, but failed to affect the functional maturation of epidermal LC. This suggests that melanoma cells participate in their own escape from immunosurveillance by preventing LC generation in the local cutaneous microenvironment.

High-performance liquid chromatography determination of bis(monoacylglycerol) phosphate and other lysophospholipids.

Bis(monoacylglycerol) phosphate (BMP) is a very minor component of the phospholipid (PL) fraction in rat uterine stromal cell cultures (U(III) cells). Under several culture conditions, including the addition of (n-3) or (n-6) polyunsaturated fatty acids, BMP selectively accumulates docosahexaenoic acid (DHA). We have recently described the structure of this PL, but its biological function is still largely unknown, except for a role in late endosomes trafficking. In order to further investigate this function, we have developed a sensitive assay for accurate determination of BMP in small biological samples. Total PL from cells, labeled or not with trace amount of [3H]DHA, were extracted and PL classes separated by thin-layer chromatography. After extraction of the gel corresponding to the BMP area, a known amount of an internal standard was added. The free hydroxyl groups of PL were totally derivatized with naproxen. Derivatized PL were separated by normal-phase high-pressure liquid chromatography and quantified using UV absorption at 231 nm. Since the sensitivity of the proposed method was about 0.1 nmol for BMP, samples of only 3 x 10(5) cells were required. The BMP level was found to be 616 +/- 46 pmol for 10(6) control cells. It was increased threefold in starved cells and significantly increased in cells cultured in the presence of exogenous phosphatidylglycerol.

Control of cell proliferation via transduction of sPLA(2)-I activity and possible PPAR activation at the nuclear level.

Pancreatic phospholipase A2 (PLA(2)-I) stimulates U(III) cells proliferation, a rat uterine cell line, after binding to membrane receptors, internalization and translocation. Here, we demonstrate that during these steps of internalization, PLA(2)-I retains its hydrolytic activity and thus could exert its proliferative effect via nuclear phospholipids hydrolysis. Since fatty acids and eicosanoids released by such activity are known to be ligands of PPAR, we study the expression of these nuclear receptors and demonstrate that, in the experimental conditions where PLA(2)-I stimulates U(III) cells proliferation, PLA(2)-I also regulates PPAR expression indicating a possible mechanism of its proliferative effect.

Involvement of calcium-independent phospholipase A2 in uterine stromal cell phospholipid remodelling.

The role of Ca2+-independent phospholipase A2 (iPLA2) in arachidonic (AA) and docosahexaenoic (DHA) acid incorporation and phospholipid remodelling in rat uterine stromal cells (UIII cells) was studied. Incorporation of AA and DHA into UIII cell phospholipids was Ca2+-independent. Bromoenollactone (BEL), a potent inhibitor of iPLA2, reduced lysophosphatidylcholine level and AA incorporation into phospholipids by approximately 20%. DHA incorporation was not affected by BEL, indicating that the pathways for AA and DHA incorporation are partially different. In control cells, the transfer of AA occurred mainly from diacyl-glycerophosphocholine (GroPCho) to alkenylacyl-glycerophosphoethanolamine (GroPEtn) and to a lesser extent from diacyl-GroPCho to diacyl-GroPEtn. [3H]DHA was redistributed from diacyl-GroPCho and alkylacyl-GroPEtn to alkenylacyl-GroPEtn. BEL treatment inhibited completely the redistributrion of AA within diacyl-GroPCho and diacyl -GroPEtn and reduced the [3H]DHA content of diacyl-GroPEtn, indicating that a BEL-sensitive iPLA2 controls the redistribution of polyunsaturated fatty acids to diacyl-GroPEtn. In contrast the redistribution of radioactive AA and DHA to alkenylacyl-GroPEtn was almost insensitive to BEL. The analysis of substrate specificity and BEL sensitivity of iPLA2 activity indicates that UIII cells exhibit at least two isoforms of iPLA2, one of which is BEL-sensitive and quite selective of diacyl species, and another one that is insensitive to BEL and selective for alkenylacyl-GroPEtn. Taken together, these results suggest that several iPLA2 participate independently in the remodelling of UIII cell phospholipids.

Hydrogen peroxide activation of Ca(2+)-independent phospholipase A(2) in uterine stromal cells.

In rat uterine stromal cells (U(III) cells), an oxidative stress induced by H(2)O(2) caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca(2+) concentration and was not inhibited by Ca(2+)-dependent phospholipase A(2) (cPLA(2)) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H(2)O(2) treatment did not impair AA esterification but significantly increased Ca(2+)-independent PLA(2) (iPLA(2)) activity. Since iPLA(2) specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H(2)O(2), we conclude that iPLA(2) activity represents the major mechanism by which H(2)O(2) increases the availability of non-esterified AA in U(III) cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H(2)O(2), suggesting a regulatory mechanism of iPLA(2) by PKC that remains to be clarified.

Bis(monoacylglycerol) phosphate in rat uterine stromal cells: structural characterization and specific esterification of docosahexaenoic acid.

In rat uterine stromal cells (U(III) cells), docosahexaenoic acid (DHA) was esterified extensively in alkenylacyl-glycerophosphoethanolamine and in an unknown phospholipid accounting for only 0.7% of the total phospholipid. The latter was identified as a bis(monoacylglycerol) phosphate (BMP) using MS. Incorporation studies using C(18:3)n-3 and C(20:5)n-3 demonstrated that BMP had a high specificity to incorporate DHA and C(22) polyunsaturated fatty acids of the (n-3) series. By contrast, polyunsaturated fatty acids of the (n-6) series were never incorporated into BMP. Incubation of U(III) cells with 5 microM DHA for 24 h increased the DHA content of BMP from 36 to 71% of the total acyl chains. [(3)H]DHA-labelled BMP purified as a single TLC spot was resolved into three peaks using HPLC. These peaks were also observed when cells were labelled with [(3)H]phosphatidylglycerol, an exogenous BMP precursor, and with [(33)P]P(i). Electrospray MS of BMP from control cells showed that the first two peaks contained the same molecular species (mainly C(22:6)n-3/C(22:6)n-3 and C(18:1)n-9/C(22:6)n-3) while the third peak mainly contained the C(18:1)n-9/C(18:1)n-9 species. The stereoconfiguration analysis of the compounds revealed an sn-glycero-3-phospho-1'-sn-glycerol configuration for the first peak and sn-glycero-1-phospho-1'-sn-glycerol configurations for the other two. BMP from rat testis was used to establish the positions of the acyl groups. More than 70% of its acyl chains were C(22:5) n-6. It was separated on HPLC into three peaks that co-migrated with the three peaks of BMP from U(III) cells. Lipase activity and NMR analysis of the second peak showed that fatty acids esterified the primary alcohol group on each glycerol moiety. We conclude that the three peaks are stereoisomeric compounds with different acyl-chain locations and may be the result of different metabolic fates depending on subcellular localization.

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood-brain barrier endothelial cells in vitro.

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.

Protein kinase C inhibitors stimulate arachidonic and docosahexaenoic acids release from uterine stromal cells through a Ca2+-independent pathway.

The mechanisms underlying arachidonic acid (AA) release by uterine stromal (U(III)) cells were studied. Stimulation of AA release by calcium ionophore and PMA are inhibited by various PKC inhibitors and by calcium deprivation. These results suggest the involvement of an AA-specific cPLA2 as the release of docosahexaenoic acid (DHA) from prelabelled cells is much lower than the release of AA. The results also show a more original stimulation of AA and DHA release induced by PKC inhibitors, which is insensitive to calcium deprivation. This stimulation is not due to acyltransferase inhibition, suggesting the participation of a Ca2+-independent PLA2 (iPLA2). However, iPLA2 activity measured in U(III) cells is inhibited by the specific iPLA2 inhibitor, BEL, and is not stimulated by PKC inhibitors, in contrast with the AA and DHA release. It seems therefore that this iPLA2 cannot be involved in this mechanism. The participation of another iPLA2, BEL-insensitive, is discussed.

Nuclear location of PLA2-I in proliferative cells.

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

Basal concentrations of free and esterified monohydroxylated fatty acids in human blood platelets.

Monohydroxylated fatty acids (HO-FA), namely 12-hydroxyeicosatetraenoic and 12-hydroxyheptadecatrienoic acids, are enzymatically formed in response to platelet activation. Different techniques, including gas chromatography (GC) and liquid chromatography-mass spectrometry (LC-MS), have been described to measure HO-FA in activated cells, but they are not well-adapted to resting cells. Measurements of free and esterified HO-FA at basal concentration require the prevention of platelet activation. For this purpose, such an activation was minimized by adding various inhibitors to the anticoagulant. Platelet recovery was greater in the protected group than in controls (473 x 10(9) +/- 4.0 x 10(9) platelets/L vs 410 x 10(9) +/- 4.53 x 10(9) platelets/L, respectively) (mean +/- SEM, n = 9, P < 0.05). Lipids were extracted and immediately hydrogenated to avoid fatty acid autoxidation occurring during the workup. Unesterified and esterified HO-FA were analyzed by GC-MS, and the former were lower in the protected group (1.52 +/- 0.84 pmol/10(9) platelets) than in the unprotected one (12.63 +/- 10.52 pmol/10(9) platelets) (mean +/- SEM, n = 9, P < 0.05). Interestingly, only traces of HO-FA were detected in both the triglyceride and sterol ester fractions, and they were also weakly esterified in phospholipids.

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells.

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.

Prolactin up-regulates prostaglandin E2 production through increased expression of pancreatic-type phospholipase A2 (type I) and prostaglandin G/H synthase 2 in uterine cells.

Uterine stromal cells produce and release PGE2, both processes being regulated by hormonal factors. In this study, we examined the effect of PRL on the PGE2 production and release measured by radioimmunoassay. For this purpose, we used a rat uterine stromal cell line, UIII cells, which produce PGE2 and contain PRL receptors. The expression of sPLA2I and PGHS (PGHS1 and PGHS2), enzymes required for PGE2 production, was also estimated by immunocytochemistry and 'Western blotting' in response to PRL. PRL (10 to 60 ng/ml) significantly increased the PGE2 release (up to 6-fold) and production, in a dose-dependent manner. Results show that PGHS1 and PGHS2 are both expressed constitutively in the uterine UIII cells, although PGHS2 is expressed at a low level. PRL did not increase PGHS1 expression, but stimulated the expression of sPLA2I and PGHS2 by 3.5- and 2.5-fold, respectively. These data show for the first time a regulation of sPLA2I and PGHS2 expression by PRL and may indicate that, in uterine cells, PRL enhances the PGE2 release and production by increasing the expression of both sPLA2I and PGHS2.

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells.

Rat uterine stromal cells (U(III)) express pancreatic type PLA2 (PLA2-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA2-I. There is a dramatic decline in PLA2-I binding in U(III) cells as they progress from a non-confluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA2-I changed with the alteration in binding, suggesting that regulation in the PLA2 binding capacity may have important implications in growth control mechanisms.

Biogenesis and metabolic fate of docosahexaenoic and arachidonic acids in rat uterine stromal cells in culture.

To gain some insight into the mechanisms involved in the opposing effects of arachidonic acid and docosahexaenoic acid on the growth of rat uterine stromal cells (UIII cells), the dynamics of the uptake, conversion, and incorporation of labeled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3), and 22:6(n-3) into lipid pools and phospholipid subclasses were examined. A very active and time-dependent conversion of [14C]18:3(n-3) to higher homologs was observed; 64.7 +/- 0.7 and 11.5 +/- 0.4% of the [14C] radioactivity incorporated in cellular lipids was recovered as 22:5(n-3) and 22:6(n-3) after 72 h incubation, respectively. The distribution of labeled fatty acids obtained after 72 h incubation with [3H]20:5(n-3) was not significantly different from that observed with 18:3(n-3). Arachidonic acid was the major fatty acid formed from [14C]18:2(n-6) and only trace amounts of 22:5(n-6) were detected. When cells were incubated for 72 h with 20:4(n-6), more than 75% of the radioactivity was recovered as arachidonate and slightly higher amounts of 22:4(n-6) and 22:5(n-6) were formed compared to those obtained after incubation with 18:2(n-6). Using both [14C]- and [3H]22:6(n-3), no significant retroconversion of labeled 22:6(n-3) occurred in the cells. More than 90% of labeled 20:4(n-6) and 22:6(n-3) taken up by the cells were esterified into phospholipids, but significant differences in their distribution among phospholipid classes and subclasses were observed. Docosahexaenoic acid was more rapidly and efficiently incorporated into phosphatidylethanolamine than 20:4(n-6) and was principally recovered in plasmalogens. Arachidonic acid was mainly incorporated in the diacyl subclasses of phosphatidylcholine and phosphatidylethanolamine and in phosphatidylinositol. The divergent profiles of these two fatty acids within the phospholipid compartments provide some information for the mechanisms of their opposite effects on UIII cell growth.

Docosahexaenoic acid is a potent inhibitor of rat uterine stromal cell proliferation.

The effect of different families of fatty acids on the proliferation of rat uterine stromal cells (UIII) was studied. Docosahexaenoic acid (DHA) exerted a strong and dose-dependent inhibitory effect (IC50 approximately 2 microM), whereas arachidonic acid (AA) stimulated UIII cell proliferation at the optimal concentration of 10 microM. Oleic, linoleic and linolenic acids were ineffective from 0.1 to 10 microM. The inhibitory effect of DHA was independent of the eicosanoid biosynthesis and lipid peroxidation, since it was not reversed by the addition of the antioxidant BHT and no significant production of oxidized species from DHA occurred in our culture conditions.

Regulation of quail oviduct phospholipase A2 activity by estradiol.

The phospholipase A2 (PLA2) activity was measured in the oviduct of immature and estradiol benzoate (EB)-treated quails. The pH profiles demonstrate the presence of two PLA2 isoforms in the avian oviduct: a neutral isoform, optimally active at pH 7-7.5 and calcium independent, responsible for most of the hydrolytic activity in the immature oviduct and poorly stimulated by estradiol; and an alkaline isoform, optimally active at pH 8-9.5 and calcium dependent, with little activity in the immature tissue but markedly stimulated by EB. After EB injection, PLA2 activation occurs at first during the prereplicative period of oviduct cells (+172% at 6 h), it is dose dependent from 0.01 to 1 mg/kg EB and can be prevented by cycloheximide together with ornithine decarboxylase activation. Moreover, estradiol was inactive on cell-free extracts of immature oviducts. These results suggest that EB increases PLA2 activity through gene activation and de novo protein synthesis. The correlation between the early stimulation of PLA2 activity and the proliferation of oviduct cells is discussed.

Oestradiol-induced changes in the composition of phospholipid classes of quail oviduct: specific replacement of arachidonic acid by docosahexaenoic acid in alkenylacyl-glycerophosphoethanolamine.

The phospholipid composition and the molecular species of the major subclasses of ethanolamine and choline glycerophospholipids were determined during the natural or oestradiol-induced development of the quail oviduct. The phospholipid concentration increased significantly during oviduct development, and the proportion of ethanolamine glycerophospholipids (EPL) remained constant while that of choline glycerophospholipids increased. The immature oviduct contained the majority of its endogenous arachidonic acid mass (71%) in EPL, mainly in alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) (49% of the total). Oestrogen treatment induced the depletion of 20:4,n-6 specifically from this pool, which indicates the biological importance of 20:4,n-6 molecular species in alkenylacyl-GPE as substrates for the oviduct phospholipases activated by oestradiol, and suggests that this EPL subclass is involved in the oestrogen-induced cell proliferation. Another striking result was the marked increase in 22:6,n-3 EPL molecular species following the oestradiol treatment and more particularly the strict substitution of 20:4,n-6 by 22:6,n-3 in alkenylacyl-GPE. We speculate that alkenylacyl-GPE molecular species containing 22:6,n-3 may participate in the arrest of oestrogen-induced proliferation.

Prostaglandin E2 production by uterine stromal cell line UIII: regulation by estradiol and evidence of an ethanol action.

We have recently established a uterine stromal cell line (UIII). The purpose of the present study was to determine whether these cells have retained the ability to produce and release prostaglandins after several passages and whether this production was regulated. UIII cells, grown in basal conditions, released a very low amount (40.6 +/- 2.9 pg/24h/10(6) cells) of prostaglandin E2 (PGE2) though cellular content was more elevated (192 +/- 23 pg/10(6) cells). Ethanol increased the cellular content but decreased the release of PGE2, whereas estradiol 17 beta (E2) increased it in a dose-dependent manner, but had no effect on the cellular content. The PGE2 release by cells grown in medium containing 10 microM arachidonate (AA) reached 1.39 +/- 0.05 ng/24h/10(6) cells, and was further increased to 2.1 +/- 0.1 ng/24 h/10(6) cells by the addition of ethanol. Under the latter condition, E2 was ineffective. This study also showed that UIII cells expressed an immunoreactive pancreatic type 14 kD PLA2. A substantial increased 14 kD PLA2 expression was observed in ethanol-treated cells, suggesting that ethanol-effect on prostaglandin production might be partly mediated by PLA2 increase. Medium supplementation with arachidonate also resulted in a significant increase of intracellular 14 kD PLA2 expression. The present results showed that uterine stromal UIII cells have retained the enzymatic machinery to produce PGE2. Moreover these data demonstrate that ethanol and E2 affect differently uterine PGE2 production.

Fluorimetric studies of calmodulin interactions with antiestrogens.

Recent cumulative data have shown that tamoxifen and its metabolites inhibit the activation of cAMP phosphodiesterase by calmodulin (CaM). In this study, the interaction of antiestrogens with CaM was investigated using a hydrophobic fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Tamoxifen (TAM) enhanced the fluorescence of TNS bound to CaM and shifted the emission maximum to lower wavelengths. These effects were concentration-dependent. No change in the apparent affinity of TNS for CaM was noted in the presence of TAM. These results suggest that TAM bound to CaM at sites distinct from those of TNS and induced a change in TNS environment. Interaction of TAM metabolites with CaM depended on the degree of alteration of the dimethylaminoethoxy side-chain. Thus, N-desmethylation or N-di-desmethylation notably reduced the interaction of the drug with the macromolecule by 24 and 77% respectively. Side-chain deamination to the primary alcohol (metabolite Y) totally suppressed the interaction. The ability of these different metabolites to interact with CaM correlates with their efficiency to inhibit CaM-dependent cAMP phosphodiesterase and their growth inhibitory potency reported previously.