Changes in serum insulin-like growth factor binding protein-2, -3, and -6 levels in patients with chronic renal failure following renal transplantation.

The pathophysiological roles of insulin-like growth factor binding protein (IGFBP)-6 have not been elucidated. Recently, we measured serum IGFBP-6 by Western immunoblot (WIB) and have found that serum IGFBP-6 levels increased in patients with chronic renal failure (CRF). In the present study, serum IGFBP-6 levels were measured in 10 patients with CRF before and 1, 7 and 14 days after renal transplantation to investigate further clinical significance and regulation of serum IGFBP-6. IGFBP-2 and -3 levels, usually elevated in patients with CRF, were also measured after renal transplantation. Serum IGFBP-2 and -6 levels from patients with CRF by Western immunoblot increased to 230+/-90% (mean +/- SD), and 400+/-110% of the reference serum, respectively, and these levels did not change after hemodialysis. Serum IGFBP-6 levels decreased to 47+/-20% of the basal level 1 day after renal transplantation, and the IGFBP-6 levels in two patients whose renal function worsened again due to rejection increased to more than 60% of the basal levels on the 14th day. In contrast to IGFBP-6, serum IGFBP-2 levels did not decrease during the 14 days after renal transplantation in all patients. Serum IGFBP-3 levels were significantly higher in CRF than normal sera (5.5+/-1.2 vs 3.7+/-0.5 microg/ml, P < 0.01), and the levels decreased to the normal range (2.7+/-1.0 microg/ml) within 1 day after the transplantation, whereas the levels increased again in one of two patients with poorly-functioning graft. In addition, we demonstrated IGFBP-6 in urine from normal adults. These results indicate that IGFBP-6 might be excreted by the kidneys and serum IGFBP-3 and -6 levels might be related with renal function, and that the regulation of serum IGFBP-2 levels differs from those of IGFBP-3 and -6.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

17 beta-estradiol increases calcium content in fetal mouse parietal bones cultured in serum-free medium only at physiological concentrations.

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17 beta-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 x 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3-4 mmol/L), calcium (1-1.25 mmol/L), insulin (6 micrograms/ML), and transferrin (6 micrograms/mL) for 4-5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 +/- 4.6 micrograms/bone [mean +/- SD], n = 50) increased by 15-20 micrograms during 4-5 days of culture. 17 beta-Estradiol increased the calcium content significantly at 10(-12) to 10(-11) mol/L, but not at lower (10(113) mol/L) or higher (10(-10) to 10(-9) mol/L) concentrations. 17 alpha-Estradiol had no effect. The stimulatory effect of 17 beta-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17 beta-estradiol was elicited not only in bones from females but also in those from males. 17 beta-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17 beta-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10(-12)-10(-11) mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17 beta-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells.

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.

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.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

[The effects of recombinant human growth hormone (r-hGH, SM-9500, genotropin) on growth failure in children with uremia. Japanese Multi-Center Study (Genotropin) Group on Children with Renal Disease].

We treated 90 pediatric patients with chronic renal failure with recombinant growth hormone (r-hGH) for 12 months to improve their growth retardation due to uremia. They were divided into two groups, non-dialyzed and dialyzed children. The dose of r-hGH was 0.5 or 1.0 IU/kg/week in dialyzed children. After 12 months of the treatment using r-hGH, growth velocity was significantly increased in any group of children. Growth velocity was stimulated to about twice as much as before treatment (that were: in non-dialyzed group, 4.2 +/- 2.6cm/year vs. 6.2 +/- 2.0cm/year, P < 0.05, in dialyzed children treated with 0.51U of r-hGH: 2.7 +/- 1.8cm/year vs. 5.2 +/- 2.6cm/year, P < 0.001, and in dialyzed children treated with 1.01U of r-hGH: 3.0 +/- 1.5cm/year vs. 6.3 +/- 2.2cm/year, P < 0.001). No severe side effects was noted and no disturbance of renal function. Our results were consistent with those reported from Europe and USA. We conclude that r-hGH treatment is very effective in improving retarded growth in children with renal disease.

[The effects of recombinant human growth hormone (r-hGH, SM-9500, genotropin) on growth disturbance in children after renal transplantation. Japanese Multi-Center Study (Genotropin) Group on Children with Renal Disease].

We treated 27 children with renal transplantation who showed growth failure due to deteriorated graft function and/or corticosteroids therapy with recombinant growth hormone (r-hGH) to improve their growth disturbance. The dose of rhGH was either 0.5 or 1.0 IU/kg/week. After 12 months treatment of r-hGH, growth velocity was significantly increased in both groups. Growth velocity was improved from 5.0 +/- 2.9 cm/year to 7.7 +/- 2.3 cm/year, P < 0.05, in 0.51U group and 3.7 +/- 2.4 cm/year to 6.3 +/- 3.3 cm/year, P < 0.05, in 1.01U group. The most important side effect of r-hGH was relevant to graft function. 7 out of all 27 children showed deterioration of graft function. However only 2 children showed significant decreases in their graft function during r-hGH therapy. Thus we conclude that r-hGH therapy was effective to improve growth failure in uremic children even after renal transplantation due to poor graft function and/or corticosteroids therapy.

Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles.

Using a highly sensitive bioassay for TSH, in which human thyroid follicles incorporate 125I and release de novo synthesized thyroid hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human thyroid follicles is too weak to cause hyperthyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperthyroidism when produced excessively.

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.

Long-term effects of growth hormone treatment on height in Turner syndrome: results of a 6-year multicentre study in Japan. Committee for the Treatment of Turner Syndrome.

Patients with Turner syndrome have many somatic characteristics, including short stature. We report the results of a 6-year multicentre clinical trial of recombinant human growth hormone (GH) therapy in 63 patients with Turner syndrome. Twenty-six patients received GH at a dose of 0.5 IU/kg/week, while 37 received GH, 1.0 IU/kg/week, by daily subcutaneous injection. At the start of GH treatment, there was no significant difference between the two groups in chronological age, bone age, height or growth rate. Both treatment groups showed a significant growth increase during treatment. The current mean height of the 12 patients over the age of 16 treated with GH, 0.5 IU/kg/week, is 145.1 +/- 4.7 cm, and in the 16 patients treated with GH, 1.0 IU/kg/week, is 144.0 +/- 2.2 cm. In conclusion, treatment with GH does increase final height in patients with Turner syndrome. However, further studies are needed to determine the optimum age for the initiation of GH therapy, the best dose regimen and the optimal time and manner of sex and anabolic steroid use.

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.

Stimulation of interleukin-4 of cell proliferation and mRNA expression of alkaline phosphatase and collagen type I in human osteoblast-like cells of trabecular bone.

Interleukin-4 (IL-4) potently inhibits bone resorption by preventing the differentiation of osteoclast precursors to osteoclasts. To elucidate the role of IL-4 in bone formation, we studied the effects of human IL-4 on human osteoblast-like cells obtained from trabecular bone, which showed increased osteocalcin production in response to 1,25-(OH)2D3 in more than 10 passages. IL-4 stimulated the proliferation of osteoblast-like cells in a concentration-dependent manner, showing the minimal and maximal stimulatory effects at 10 pg/ml and 100-1000 pg/ml, respectively. IL-4 also stimulated the expression of alkaline phosphatase mRNA (1.7-fold) and the enzyme activity to the same extent at 10-100 pg/ml. Furthermore, IL-4 stimulated collagen type I mRNA expression in human osteoblast-like cells. The cytokine did not affect osteocalcin production in a short culture period (3 days). These in vitro findings suggest that IL-4, a bone-resorption-inhibitory cytokine produced by activated T cells in bone marrow, may exert an anabolic effect on osteoblast-like cells in trabecular bone through a paracrine mechanism.

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)

Octreotide treatment results in the inhibition of GH gene expression in the adenoma of the patients with acromegaly.

Seven patients with growth hormone (GH)-secreting pituitary adenoma were treated preoperatively with octreotide (Sandostatin or SMS 201-995; a somatostatin analogue), and were compared with 18 non-treated patients in their clinical courses and adenoma analyses. Octreotide treatment improved the endocrinological data in all 7 cases. The octreotide-treated adenomas were soft and easily removed by suction and curettage. The postoperative normalization of endocrinological data was encountered more often in the octreotide-treated cases than in the non-treated, although the statistical significance was not observed by the limited number of cases. The adenoma tissues were examined with conventional histology and immunohistochemistry, and the amount of GH messenger ribonucleic acid (mRNA) was quantitatively assessed. The studies demonstrated: 1) No fibrosis nor necrosis was observed in the adenomas from the octreotide-treated patients. 2) Immunohistochemistry for human GH revealed no remarkable differences between the octreotide-treated and the non-treated adenomas. 3) The amounts of GH mRNA in the adenoma from the octreotide-treated patients were 4.2 +/- 1.8 (mean +/- SEM; expressed in an arbitrary unit) and were significantly less than those from the non-treated (33.6 +/- 9.1). These data suggest that octreotide inhibits not only GH release from the adenoma but also its biosynthesis.

Insulin-like growth factor I receptors and insulin-like growth factor-binding proteins in human parathyroid tumors.

Studies have demonstrated the presence of epidermal growth factor receptors in human parathyroid tumors. However, there is little information on the effect of other peptide growth factors on parathyroid cell growth. We therefore studied the interaction of insulin-like growth factor I (IGF-I) with human parathyroid tumor cells. Parathyroid tissues were obtained from 24 patients with primary or secondary hyperparathyroidism. There were 15 solitary adenomas, 5 carcinomas, and 4 hyperplastic tissues. First, the binding of [125I]IGF-I to the crude membrane fractions was studied by competitive inhibition with unlabeled IGF-I. Second, isolated parathyroid cells were cultured with IGF-I and examined for DNA synthesis. The IGF-binding protein (IGFBP) content of tissue homogenates was determined by ligand blot analysis. The binding of [125I]IGF-I to parathyroid membranes was dependent on time, temperature, and pH of the medium. Maximum binding was obtained after incubation for 18 hours at 4 degrees C. Specific binding to parathyroid cancer membranes (mean +/- SE, 10.75 +/- 10.55%/mg protein) was significantly (p < 0.05) greater than that in adenoma tissues (3.71 +/- 2.11%/mg). The value in hyperplastic tissues (4.78 +/- 2.97%/mg) was not different from that in adenomas. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Cultured parathyroid cells responded to IGF-I with increased DNA synthesis. The parathyroid tumor tissues expressed IGFBPs. These results suggest that IGF-I and IGFBPs are involved in the growth regulation of parathyroid tumor cells.

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.