Akt activation and localisation correlate with tumour invasion and oncogene expression in thyroid cancer.

INTRODUCTION: Akt activation is involved in the pathogenesis of inherited thyroid cancer in Cowden's syndrome and in sporadic thyroid cancers. In cell culture, Akt regulates thyroid cell growth and survival; but recent data suggest that Akt also regulates cell motility in non-thyroid cell lines. We therefore sought to evaluate the role of Akt in thyroid cancer progression. METHODS: We evaluated 46 thyroid cancer, 20 thyroid follicular adenoma, and adjacent normal tissues samples by immunohistochemistry for activated Akt (pAkt), Akt 1, 2, and 3, and p27 expression. Immunoblots were performed in 14 samples. RESULTS: Akt activation was identified in 10/10 follicular cancers, 26/26 papillary cancers, and 2/10 follicular variant of papillary cancers, but in only 4/66 normal tissue samples and 2/10 typical benign follicular adenomas. Immunoactive pAkt was greatest in regions of capsular invasion; and was localised to the nucleus in follicular cancers and the cytoplasm in papillary cancers, except for invasive regions of papillary cancers where it localised to both compartments. Immunoactive Akt 1, but not Akt 2 or Akt 3, correlated with pAkt localisation, and nuclear pAkt was associated with cytoplasmic expression of p27. In vitro studies using human thyroid cancer cells demonstrated that nuclear translocation of Akt 1 and pAkt were associated with cytoplasmic p27 and cell invasion and migration. Cell migration and the localisation of Akt 1, pAkt, and p27 were inhibited by PI3 kinase, but not MEK inhibition. DISCUSSION: These data suggest an important role for nuclear activation of Akt 1 in thyroid cancer progression.

Structural investigations of a new familial dysalbuminemic hyperthyroxinemia genotype.

BACKGROUND: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously. METHODS AND RESULTS: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 x 10(-6), 2.64 x 10(-7), and 2.49 x 10(-7) mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein. CONCLUSIONS: The K(d) values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the K(d) values determined in this study. Possible explanations for these discrepancies are discussed.

Growth hormone directly inhibits leptin gene expression in visceral fat tissue in fatty Zucker rats.

Growth hormone (GH) is known to interact with adipose tissue and to induce lipolysis. Adipocytes produce leptin which regulates appetite and energy expenditure. In order to elucidate the role of GH in leptin production, we studied the effect of GH on leptin gene expression and body fat in fatty Zucker rats, a model of obesity with resistance to both leptin and insulin. Recombinant human GH administered subcutaneously at 0.5 mg/kg per day (low dose) as well as at 1.65 mg/kg per day (high dose) reduced leptin mRNA levels in epididymal fat tissue but not in subcutaneous fat tissue after 7 days. GH administration only at the high dose reduced percentage body fat. Insulin-like growth factor-I infusion (200 microg/kg per day) did not change percentage body fat or leptin mRNA levels in epididymal fat. These observations suggest that GH directly interacts with adipose tissue and reduces leptin gene expression in visceral fat tissue.

Serum leptin levels and bioelectrical impedance assessment of body composition in patients with Graves' disease and hypothyroidism.

We investigated whether thyroid status modulates serum leptin concentrations and body composition as determined by bioelectric impedance analysis (BIA). The percent body fat mass (%FM) in male Graves' disease was significantly lower than that in age- and sex- matched normal subjects, at the levels of 11.4+/-6.4% (mean+/-SD) vs 19.9+/-9.2% for men (n=12, P<0.05) but not for women (22.6+/-7.6% vs 24.9+/-13.1%, n=28). In contrast, in female hypothyroidism (n=11) %FM was significantly higher than that in normal subjects (32.9+/-11.5%, P<0.01). Among other body composition parameters, the percentage of body water (%BW), and lean body mass (LBM) were significantly lower in hypothyroid patients, and the ECM (extracellular mass)/BCM (body cell mass) ratio was significantly (P<0.0001) increased in Graves' disease which was the result of marked depletion of BCM with concomitant expansion of ECM. The serum leptin levels were significantly decreased in male Graves' patients (2.3+/-0.7 ng/ml, P<0.05), whereas in female Graves' patients (8.8+/-5.9 ng/ml) and patients with hypothyroidism (9.5+/-7.6 ng/ml), the levels were not different from those of normal controls matched for BMI or %FM. There was a positive correlation between serum leptin levels and %FM in female Graves' patients (r=0.635, P=0.001) and in hypothyroid patients (r=0.801, P=0.014) but not in male Graves patients. There was no significant relationship between serum leptin levels and thyroid hormones, TRAb, or TSAb. In euthyroid obese subjects there was a positive relationship between serum leptin levels and serum TSH levels (r=0.37, P<0.01). These results suggest that hyperthyroidism is characterized by the decreased fat mass and serum leptin levels in men, but female patients appear to be resistant to the effect of thyroid hormones. Together with previous reports, thyroid status has a minor role in the regulation of serum leptin levels.

Interaction between leptin and growth hormone (GH)/IGF-I axis.

In order to identify the mutual interaction between GH and leptin, we studied the effect of GH on fatty Zucker rats. GH administration at a high dose (5.0 IU/kg) reduced % body fat after 7 days. The leptin mRNA level in subcutaneous fat tissue was not changed but that in epididymal fat tissue was decreased by an even lower dose of GH (1.5 IU/kg). IGF-I treatment (200 microg/kg/day) did not change the % body fat or leptin mRNA level. These observations suggest that GH directly interacts with visceral fat and reduces fat mass and leptin expression. We also measured serum leptin levels in patients. The levels in patients with acromegaly were significantly lower than those in normal subjects with the same amount of body fat, but serum IGF-I and urinary C peptide excretion rates were higher in the acromegalic. These observations also suggests that GH directly interacts with adipose tissue and reduces leptin expression. Next we investigated the direct action of leptin on GH release from the pituitary. Leptin pretreatment of pituitary cells in culture or rats in a fasted or fed condition did not change GRH induced GH secretion. As indicated also by other recent studies, leptin may increase GRH or decrease somatostatin secretion by the hypothalamus. Thus GH interacts with fat tissues and leptin may be a good marker of the interaction.

Effect of cytokines on production of insulin-like growth factor binding proteins (IGFBPs) from human fibroblasts in culture.

Bioactivity of Insulin-like growth factors (IGEs) are positively or negatively regulated by IGF binding proteins (IGFBPs). Like IGFs, production of IGFBPs are influenced by a number of hormonal factors. We studied the effects of cytokines on production of IGFBPs in human fibroblasts in culture. Both IL-1beta and TNF-alpha inhibited IGFBP-3 (42/38 KDa species) production in a concentration dependent manner judged by Western ligand blot. Expression of IGFBP-3 mRNA was also decreased by these cytokines. Moreover, the treatment with IL-1beta and TNF-alpha resulted in appearance of smaller mol weight (26 KDa) immunoreactive IGFBP-3 fragment(s) which lacked the ability to bind 125I-IGFs, indicating that these cytokines degrade IGFBP-3 via activation of proteases. Both IL-1beta and TNF-alpha decreased production of IGFBP-4, whereas they increased that of IGFBP-6, IL-6 had little effect on production of IGFBPs. Likewise, interferon-gamma failed to affect of production of IGFBPs except at high concentrations. The present data demonstrate that cytokines, especially IL-1beta and TNF-alpha are potent regulators of IGFBPs production and degradation. These cytokines may alter tissue uptake of IGFs and help to counteract the catabolic state induced by them.

High prevalence of T354P sodium/iodide symporter gene mutation in Japanese patients with iodide transport defect who have heterogeneous clinical pictures.

A missense and loss of function mutation of the Na+/I- symporter (NIS) gene, T354P [Thr354-->Pro (ACA-->CCA)], was found in the homozygous state in two unrelated Japanese patients with iodide transport defect. In this study we have identified the homozygous T354P NIS germline mutation in seven Japanese patients, including one previously reported, from five unrelated families. No other nucleotide changes were found in the coding regions and the exon-intron boundaries of the NIS gene in these seven patients. These results suggest a common prevalence of the T354P mutation in Japanese patients. Although these seven patients have the identical NIS mutation, T354P, marked heterogeneity in clinical pictures, especially concerning goiter and hypothyroidism, were noted among them. Therefore, another factor(s), but not the nature of the NIS mutation, may account for the clinical heterogeneity among patients with the iodide transport defect. We have previously reported that the NIS messenger ribonucleic acid was markedly increased in the thyroid of a patient with the homozygous T354P mutation. In this study we demonstrated that the NIS proteins in the patients' thyroids were significantly increased (approximately 10-fold) by Western blot analysis of integral membrane proteins using an antibody against the C-terminal peptide of the human NIS. Furthermore, we showed by immunohistochemical staining that the T354P mutant NIS proteins were overexpressed in the basal and lateral plasma membranes of patients' thyrocytes.

Effect of growth hormone (GH) on serum concentrations of leptin: study in patients with acromegaly and GH deficiency.

As leptin, an ob gene product, plays a pivotal role in the regulation of adiposity and energy homeostasis, the level of its expression is likely to fluctuate under various physiological, nutritional, and disease conditions. Reports regarding the effect of GH on serum leptin levels are inconsistent. We have measured serum leptin levels and correlated them with several variables in patients with acromegaly, patients with adult GH deficiency (GHD), and normal controls. In 116 normal subjects, the mean serum concentration of leptin was 5.0+/-2.8 (mean +/- SD) ng/mL in men (n = 42) and 10.7+/-7.3 ng/mL in women (n = 73), respectively. As reported previously, the leptin levels in women were significantly (P < 0.001) higher than in men, and there was a strong positive correlation between log-transformed serum leptin levels and percent body fat in simple regression analysis (in men: r = 0.606; P < 0.0001; in women: r = 0.707; P < 0.0001). In 36 acromegalic patients, the percent body fat mass was significantly lower than that in normal subjects, and the mean serum leptin level was 2.2+/-1.8 ng/mL in men (n = 18) and 3.6+/-2.5 ng/mL in women (n = 18). Analysis of covariance revealed that serum leptin levels in acromegalics were significantly lower than those in normal subjects after correcting percent body fat (P = 0.016 for men and P < 0001 for women). In male patients with GHD (n = 20), the mean percent body fat was significantly (P < 0.05) higher than that in age-matched controls, whereas the value in female GHD patients (n = 15) did not differ from that in age-matched controls. Serum leptin levels in GHD patients were 5.1+/-2.5 ng/mL in men and 11.5+/-8.1 ng/mL in women, which were not different from those in normal subjects adjusted for percent body fat mass. In multiple regression analysis models with log-transformed leptin as the dependent variable, gender, percent body fat (or body fat mass), and serum insulin-like growth factor I levels entered the equations at a statistically significant level. These data suggest that excess GH/insulin-like growth factor I reduces serum leptin levels by reducing body fat mass and/or by unknown mechanisms.

Oncostatin M: a new potent inhibitor of iodine metabolism inhibits thyroid peroxidase gene expression but not DNA synthesis in porcine thyroid cells in culture.

The functions of thyroid cells are regulated by a number of cytokines and growth factors in addition to TSH. Recent studies have revealed that several cytokines including interleukin (IL)-6 are involved in thyroid dysfunction. Oncostatin M (OSM) is a glycoprotein belonging to the same family of cytokines as IL-6, to which it is related by sequence and structural homology and the use of the signal-transducing receptor component gp130. We, therefore, studied the effect of OSM on iodide uptake and DNA synthesis by porcine thyroid cells in culture. OSM increased c-fos and c-jun mRNA levels but did not stimulate DNA synthesis. OSM inhibited iodide uptake stimulated by TSH; while IL-6 also inhibited iodide uptake, it was only about one-tenth as potent. IL-6 had about the same potency as OSM when it was added with soluble IL-6 receptor. OSM had no effect on cAMP production but inhibited iodide uptake stimulated by 8-bromo-cAMP and forskolin. These findings suggest that OSM exerts its inhibitory effects at the post-cAMP production step(s). OSM also inhibited thyroid peroxidase mRNA levels but had little effect on thyroglobulin mRNA levels. Investigations of the signal transduction system showed that gp130 and leukemia inhibitory factor (LIF) receptor beta subunit mRNA were detectable in porcine thyroid cells by reverse transcription (RT)-polymerase chain reaction (PCR). Together with the report that serum OSM and IL-6 concentrations are elevated to the same levels in patients with sepsis, these results suggest that OSM may contribute to the thyroid dysfunction in this condition.

Degradation of cell surface heparan sulfates decreases the high affinity binding of basic FGF to endothelial cells, but not to FRTL-5 rat thyroid cells.

The role of cell surface heparan sulfate proteoglycans in the effect of basic fibroblast growth factor (bFGF) on FRTL-5 rat thyroid cells was investigated and compared with that of endothelial cells. FRTL-5 cells were incubated for 2 h with heparitinase (0.5-5.0 mU/mL), which specifically degrades heparan sulfate proteolgycans, and then stimulated by bFGF. The mitogenic effect of bFGF was estimated by measuring [3H]thymidine incorporation. Although cell surface heparan sulfates have been believed to be necessary for bFGF binding to its high affinity receptors, the heparitinase treatment had no significant effect on the DNA synthesis of FRTL-5 cells stimulated by bFGF. The binding study revealed that heparitinase treatment decreased low affinity bindings of [125I]bFGF to FRTL-5 cells by only 50% and did not attenuate the high affinity binding, while the same treatment abolished the high and low affinity binding to bovine pulmonary artery endothelial (CPAE) cells. Analysis of trypsin accessible cell surface 35SO4-labeled materials by Q-sepharose anion-exchange column chromatography showed that heparan sulfate proteoglycans, peaked at 0.55 M NaCl elution, disappeared from the surface of FRTL-5 cells after treatment with 2.0 mU/mL of heparitinase, indicating that the heparitinase resistant low-affinity binding sites are not heparan sulfates. These results demonstrate that cell surface heparan sulfates are not required for the high affinity binding of bFGF to FRTL-5 rat thyroid cells, while proteoglycans are necessary for binding to endothelial cells, and suggest that the mechanism of the action of bFGF is different in rat thyroid cells compared with endothelial cells.

Big insulin-like growth factor II-producing hepatocellular carcinoma associated with hypoglycemia.

A 76-year-old female with a hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) was hospitalized because of fasting hypoglycemia. Her sera contained a low concentration of immunoreactive insulin and insulin-like growth factor (IGF)-I, while the IGF-II level was normal. However, most of the IGF-II consisted of the high molecular weight form (big IGF-II). The tumor tissue contained fetal type of IGF-II mRNA (6.0 kb). Furthermore, we found that one of the four patients examined with HCV-related HCC had big IGF-II in serum. This indicates that non-islet cell tumor hypoglycemia (NICTH) in HCV-related HCC might be accompanied by production of big IGF-II by the tumor.

Expression of recombinant human thyrotropin receptor in myeloma cells.

Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.

Effects of transforming growth factor alpha (TGF-alpha) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells.

Transforming growth factor alpha (TGF-alpha) is a potent mitogen that is similar structurally to epidermal growth factor (EGF). As EGF is a potent growth stimulator and an inhibitor of iodine metabolism in cultured thyroid cells of several species, we studied whether TGF-alpha has similar effects using porcine thyroid cells in culture. Recombinant human TGF-alpha dose-dependently stimulated DNA synthesis of thyroid cells, with maximal stimulation (eight- to ninefold above basal) occurring at 2 nmol/l. The potency was approximately 50% that of mouse EGF and correlated with the ability to compete with EGF for receptor binding, suggesting that the action of TGF-alpha is mediated by interaction with EGF receptors. When thyroid cells were cultured for 3 days with thyrotropin (TSH) in the presence of TGF-alpha, TSH-induced iodide uptake was inhibited in a dose-dependent manner. The potency of TGF-alpha again was approximately 50% that of EGF. Transforming growth factor alpha did not inhibit TSH-stimulated cAMP production. Moreover, iodide uptake stimulated by either forskolin or 8-bromo-cAMP also was inhibited by TGF-alpha. Thus, we conclude that TGF-alpha inhibits TSH-induced iodine metabolism largely by acting at the steps distal to cAMP production. Northern blot analysis revealed expression of TGF-alpha mRNA in porcine thyroid cells. These observations suggest that TGF-alpha acts as an autocrine modulator of growth and differentiated functions in porcine thyroid cells.

Basic fibroblast growth factor (FGF-2) in renal cell carcinoma, which is indistinguishable from that in normal kidney, is involved in renal cell carcinoma growth.

To investigate the role of basic fibroblast growth factor (FGF) in renal cell carcinoma growth, we have analyzed the expression of mRNA of basic FGF. In 7 of 15 cases, basic FGF mRNA level in renal cell carcinoma tissues was higher than that in corresponding normal tissues. However, the tumor-to-normal ratios of expression levels are chiefly less than 2.0 and, in 5 cases, are even less than 1.0. Furthermore, there was no correlation between the ratio and the clinical stage. In protein analysis, we could not find any difference between basic FGF extracted from renal cell carcinomas and that from normal kidney tissues in bioactivity, immunoreactivity, molecular weight and affinity to heparin. On the other hand, anti-basic FGF monoclonal antibody inhibited the growth of a renal cell carcinoma cell line, VMRC-RCW, and this inhibition was reversed by an extraphysiological amount of exogenous basic FGF (100 ng./ml.). These results suggest that basic FGF itself may have no pivotal role in renal cell carcinoma etiology but is involved in the growth of renal cell carcinomas in an autocrine manner.

Interaction of endothelin-1 with porcine thyroid cells in culture: a possible autocrine factor regulating iodine metabolism.

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a Kd of 0.45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of inhibitory actions of minocycline and doxycycline on ascitic fluid production induced by mouse fibrosarcoma cells.

Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5-4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HCl) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC50s of 5 microM and 8 microM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 microM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.

Inhibition of human pancreatic cancer cell (MIA PaCa-2) growth by cholera toxin and 8-chloro-cAMP in vitro.

The effects of cholera toxin (CT) and 8-chloro-cAMP (8-Cl-cAMP) on cell growth were investigated using two human pancreatic carcinoma cell lines (MIA PaCa-2, Panc-1). CT, which catalyses the ADP ribosylation of Gs, suppresses the proliferation of MIA PaCa-2(PC) cells. CT at the low dose of 0.1 pg ml-1 was inhibitory of PC cell growth, and the maximum suppression (70%) was achieved at a CT concentration of 100 pg ml-1. This phenomenon was reversible. The production of cAMP by CT (100 pg ml-1) in PC cells was enhanced 320-fold compared with the control. In addition, cAMP analogues (8-Cl-cAMP, 8-Br-cAMP) and forskolin decreased the growth rate of PC cells in a dose-dependent manner. These results support the view that CT suppresses PC cell growth by stimulating cAMP production. Conversely, Panc-1 cells were far less sensitive to CT in cell growth and cAMP production. 8-Cl-cAMP was also less effective on Panc-1 cell growth. The binding of an insulin-like growth factor (IGF)-I and transforming growth factor (TGF)-alpha, which has been shown to stimulate PC cell growth in an autocrine manner, to PC cells was not modified in cells treated with CT or 8-Cl-cAMP. The results suggest that the inhibitory actions of these substances do not occur at the level of the receptor for IGF-I or EGF/TGF-alpha. We have previously shown that phorbol esters, which decrease the binding of TGF-alpha to PC cells, has an anti-proliferative activity on these tumour cells. Inhibited cell growth by maximum suppressive dose of CT or 8-Cl-cAMP was further inhibited by TPA. In addition, an oncogene product of K-ras which is commonly activated in pancreatic cancer, was increased by CT and 8-Cl-cAMP. It is concluded that CT and 8-Cl-cAMP inhibit PC cell growth, presumably in a similar manner, and their mechanism(s) of action may be different from that of TPA. The anti-proliferative effect of CT or 8-Cl-cAMP was enhanced by TPA, implying that the combination of these substances results in increased inhibition of the PC cell growth.