Correction: Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

MUC1-C overexpression has been associated with the progression of pancreatic tumors by promoting the aggressive and metastatic phenotypes. As MUC1 is a STAT3 target gene, STAT3 plays a major role in regulating MUC1-C expression. In this study, we report an alternative mechanism by which integrin-linked kinase (ILK) post-transcriptionally modulates the expression of MUC1-C by maintaining its protein stability in pancreatic cancer cells. We found that ILK acts in concert with STAT3 to facilitate IL-6-mediated upregulation of MUC1-C; ILK depletion was equally effective as STAT3 depletion in abolishing IL-6-induced MUC1-C overexpression without disturbing the phosphorylation or cellular distribution of STAT3. Conversely, ectopic expression of constitutively active ILK increased MUC1-C expression, though this increase was not noted with kinase-dead ILK. This finding suggests the requirement of the kinase activity of ILK in regulating MUC1-C stability, which was confirmed by using the ILK kinase inhibitor T315. Furthermore, our data suggest the involvement of protein kinase C (PKC)δ in mediating the suppressive effect of ILK inhibition on MUC1-C repression. For example, co-immunoprecipitation analysis indicated that ILK depletion-mediated MUC1-C phosphorylation was accompanied by increased phosphorylation of PKCδ at the activation loop Thr-507 and increased binding of PKCδ to MUC1-C. Conversely, ILK overexpression resulted in decreased PKCδ phosphorylation. From a mechanistic perspective, the present finding, together with our recent report that ILK controls the expression of oncogenic KRAS through a regulatory loop, underscores the pivotal role of ILK in promoting pancreatic cancer progression.

Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.

Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

Met interacts with EGFR and Ron in canine osteosarcoma.

The receptor tyrosine kinase (RTK) Met is known to be over-expressed in canine osteosarcoma (OSA). In human cancers, the RTKs Met, epidermal growth factor receptor (EGFR) and Ron are frequently co-expressed and engage in heterodimerization, altering signal transduction and promoting resistance to targeted therapeutics. We found that EGFR and Ron are expressed in canine OSA cell lines and primary tissues, EGFR and Ron are frequently phosphorylated in OSA tumour samples, and Met is co-associated with EGFR and Ron in canine OSA cell lines. Transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF) stimulation induced amplification of ERK1/2 and STAT3 phosphorylation in OSA cells and Met was phosphorylated following TGFα stimulation providing evidence for receptor cross-talk. Lastly, treatment of OSA cells with combined gefitinib and crizotinib inhibited cell proliferation in an additive manner. Together, these data support the notion that Met, EGFR and Ron interact in OSA cells and as such, may represent viable targets for therapeutic intervention.

3-Phosphoinositide-dependent protein kinase-1/Akt signalling and inhibition in a canine prostate carcinoma cell line.

Deregulation of the 3-phosphoinositide-dependent protein kinase-1 (PDK-1)/Akt signalling pathway is associated with prostate cancer development and progression. Inhibition of PDK-1/Akt signalling can be achieved using structurally optimized celecoxib derivatives such as OSU-03012. In this study, we treated the novel canine prostate cancer cell line, Ace-1, with OSU-03012 or dimethyl sulphoxide in vitro. We found that Akt was constitutively phosphorylated in the canine prostate cancer cell line Ace-1 and that there was a dose-dependent decrease in cell viability, and Akt and glycogen synthase kinase-3beta phosphorylation, in response to OSU-03012 treatment. This was accompanied by a dose-dependent increase in apoptosis. These data suggest that Akt signalling pathway inhibition is a potential strategy for the treatment of dogs with prostate cancer and that canine prostate cancer is a relevant large animal model for evaluating Akt pathway inhibitors such as OSU-03012 for use in people.

17 beta-estradiol-regulated expression of protein tyrosine phosphatase gamma gene in cultured human normal breast and breast cancer cells.

BACKGROUND: Protein tyrosine phosphatase gamma (PTP gamma) has been implicated as a potential tumor suppressor gene in kidney and lung adenocarcinomas. We have previously shown that PTP gamma mRNA expression levels are lower in DES-induced kidney tumors than in normal kidneys of Syrian hamsters. The goals of the present study were to determine if PTP gamma mRNA is present in both normal and cancerous human breast cells, and to investigate the estrogenic regulation of PTP gamma mRNA expression in these cell types. METHODS: Primary cultured human breast cells derived from surgical specimens of mammoplasty and breast cancer patients, as well as human breast cancer cell lines were used for the study. RT-PCR and RNase protection assay was utilized to detect and quantify levels of PTP gamma mRNA among the cell types used and between control and 17 beta-estradiol (E2)-treated cells. Transient transfection of human estrogen receptor (ER) into MDA-MB-231 human breast cancer cells was performed to establish the role of ER in the regulation of PTP gamma mRNA expression. RESULTS: The results show that PTP gamma mRNA is expressed in primary cultured human breast cells isolated from mammoplasty and breast cancer patients, as well as in human cancer cell lines, and that E2 significantly inhibits PTP gamma expression in ER-positive human breast cancer cells via an ER-mediated mechanism. We show that PTP gamma mRNA levels are lower in human breast cancer cells than in normal human breast cells. Furthermore, we report that PTP gamma mRNA expression is inhibited by E2 in a dose-dependent manner in primary cultured breast cells. After treatment with 20 nM E2 for 24 hours, PTP gamma mRNA was significantly suppressed in primary cultured cancerous and non-cancerous cells from breast cancer patients, as well as in the ER-positive MCF-7 cell line by 50%, 85%, and 66%, respectively. In contrast, the PTP gamma mRNA expression levels did not change in similarly treated ER-negative MDA-MB-231 cells. Sensitivity to E2-induced suppression could be restored (94% inhibition) by transfecting MDA-MB-231 cells with an ER expression plasmid. CONCLUSIONS: Our results are the first to suggest that PTP gamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer which may play an important role in neoplastic processes of human breast epithelium.

The effects of gossypol on the invasiveness of MAT-LyLu cells and MAT-LyLu cells from the metastasized lungs of MAT-LyLu-bearing Copenhagen rats.

Recently, we isolated a novel subline of the MAT-LyLu cell line from the metastasized lungs of MAT-LyLu-bearing Copenhagen rats (MLL cells). In this study, we compared the MLL cells to the parental MAT-LyLu cells with respect to invasive ability, mRNA expression level for the nm23 metastasis suppressor gene, and response to gossypol (GP), a natural compound with documented antiproliferative and antimetastatic activity, in an in vitro invasion assay. ML cells were isolated from mechanically dissociated metastasized lungs from MAT-LuLu-bearing Copenhagen rats. Comparisons of the invasive ability and steady-state levels of nm23 mRNA between MLL and MAT-LuLu cells were determined by in vitro invasion assay and RT-PCR, respectively. The results show that MLL cells display a higher penetration percentage than MAT-LyLu cells in the in vitro invasion assay. Furthermore, RT-PCR revealed that MLL cells possess lower steady state levels of nm23 mRNA than MAT-LyLu cells, suggesting a molecular basis for the observed differences in in vitro invasive ability. Finally, both MLL and MAT-LyLu cells were susceptible to gossypol, which induced dose-dependent inhibition of invasive activity. These results report the isolation of a novel, more highly invasive subline of the MAT-LyLu cell line that is as susceptible to the inhibitory effects of gossypol as the parental MAT-LyLu cells. The MLL cells, in combination with the parental MAT-LyLu cells, can be valuable tools for investigating the biology and behavior of metastatic cells and their response to chemotherapeutic/preventive agents.

The (-)-enantiomer of gossypol inhibits proliferation of stromal cells derived from human breast adipose tissues by enhancing transforming growth factor beta1 production.

We report here that (-)-gossypol significantly inhibits the proliferation of stromal cells derived from human breast adipose tissues (human breast adipose stromal cells) in a dose-dependent manner. The mechanisms involved in the anti-proliferative action of (-)-gossypol on adipose stromal cells were also investigated. (-)-Gossypol stimulated transforming growth factor (TGFbeta1) secretion after 24 h, and RNase protection assay showed that TGFbeta1 mRNA levels were increased as well. We also observed that TGFbeta1 significantly inhibited the growth of human breast adipose stromal cells in a dose-dependent manner. When human breast adipose stromal cells were co-incubated with 5 microM (-)-gossypol and 50 microgram/ml of anti-TGF-beta1,-beta2,-beta3 antibody, growth inhibition caused by (-)-gossypol was completely abrogated. This study indicates that the anti-proliferative activity of (-)-gossypol on human breast adipose stromal cells may be mediated by changes in TGF 1 production.

Keratinocyte growth factor (KGF) induces aromatase activity in cultured MCF-7 human breast cancer cells.

Estrogen is the major hormonal stimulus for growth of the hormonal-dependent type of breast cancer. The rate-limiting step in the conversion of androgens to estrogens in breast tumors is catalyzed by aromatase, one of a series of related P-450 enzymes involved in the production of steroid hormones. An interesting correlation has been found between KGF mRNA and aromatase mRNA expression in human breast tumors. Tumors that express aromatase mRNA exhibit strong KGF expression, while tumors that do not express aromatase are weak or negative for KGF expression. Thus, it is reasonable to theorize that a possible association between KGF and aromatase in controlling human breast tumor growth exists. The purpose of the current study was to establish whether there is any interaction between KGF, which is known to have epithelial-specific mitogenic activity on breast cancer cells in vitro, and the synthesis of estradiol within the hormone-dependent breast cancer epithelial cells. In the present study, we have demonstrated that KGF stimulates aromatase activity in human breast cancer cells (MCF-7) in a dose-dependent manner. Our data shows that recombinant human KGF, at a dose as low as 10 ng/ml, can significantly increase aromatase activity 2-fold over controls. In agreement with this observation, we also found that aromatase mRNA levels were increased after 10 ng/ml KGF treatment in MCF-7 cells. These results indicate that the stimulatory effect of KGF on aromatase activity may be mediated by alterations in aromatase mRNA levels or in the efficiency of the translation of the message in MCF-7 cells. In addition, our results have demonstrated that modulation of aromatase activity appears to correlate with the stimulation of proliferative activity by KGF in MCF-7 cells. These results are consistent with our previous observations that estradiol-17 beta stimulates KGF expression in human breast cancer stromal cells, leading to the speculation that breast malignant transformation is associated with a positive feedback stimulation, whereby estradiol-17 beta stimulates breast cancer stromal cell production of KGF, and KGF subsequently stimulates aromatase activity in breast cancer cells, consequently raising levels of estradiol-17 beta, in turn acts on breast stromal cells to yield more KGF. Such a positive feedback loop could play an important role in the loss of growth control in human breast cancer cells.

Estrogen-induced keratinocyte growth factor mRNA expression in normal and cancerous human breast cells.

The local recurrence rate of breast cancer has been reported to be unusually high at the surgical scar. Such breast cancer recurrence is believed to be triggered by the release of growth factors into the healing wound. Observations from an animal model have also demonstrated that KGF expression is dramatically induced by creation of full thickness wounds in mouse skin. Since KGF is an epithelial cell-specific mitogen in rat mammary epithelium, it is reasonable to speculate that KGF may be also involved in regulating human breast cancer cell growth. The purpose of the present study was to determine the effect of estradiol-17 on KGF gene expression in normal human breast stromal cells, as well as in human breast cancer stromal cells, and the mechanisms by which estradiol-17 regulates breast epithelial proliferation. Our results show that KGF expression was not effected by estradiol-17 treatment in normal human breast stromal cells. In contrast, KGF expression was stimulated by estradiol-17 in human breast cancer stromal cells. KGF mRNA levels have also been examined in normal human breast stromal cells and human breast cancer stromal cells. An interesting correlation was found between KGF expression and estradiol-17 regulation in these cell types. Normal human breast stromal cells which do not response to estradiol-17 have lower KGF mRNA level than the cancer cells which KGF expression is stimulated by estradiol-17. Our data also demonstrate that recombinant human KGF significantly stimulate normal human breast and human breast cancer epithelial cell proliferation in a dose-dependent manner. Since we have shown that estradiol-17 induces KGF mRNA expression in human breast cancer stromal cells, KGF may be involved at least in part in the stimulatory pathway that is initiated by estradiol-17 in human breast cancer epithelial cells.

Gossypol arrests human benign prostatic hyperplastic cell growth at G0/G1 phase of the cell cycle.

Recently we demonstrated that gossypol (GP), a male antifertility agent, is a potent inhibitor of malignant human prostate cancer cell growth that acts by arresting cells in G0/G1 phase and that this inhibitory effect may be mediated by transforming growth factor-beta 1 (TGF-beta 1). In this study we examined the effect of GP on the growth of prostatic cells from human benign prostatic hyperplasia (BPH) patients in vitro. Consistent with its inhibitory effect on the growth of malignant human prostate cancer cells, GP also acts as a potent inhibitor of cultured human BPH cell growth as assessed by thymidine incorporation assay. These results were confirmed by flow cytometric analysis which revealed that treatment of human BPH cells with increasing concentrations of GP resulted in a dose-dependent accumulation of cells in the G0/G1 phase with a concomitant decrease in cells progressing to the S and G2/M phases. Since inhibition of prostate cancer cells by GP appears to be mediated by TGF-beta 1, we also investigated the effect of GP on TGF-beta 1 gene expression in BPH cells. The results show that GP treatment resulted in a marked elevation of TGF-beta 1 gene expression indicating that TGF-beta 1 might be involved at least in part in the inhibitory pathway that is initiated by GP.

Gossypol induces spermidine/spermine N1-acetyltransferase in canine prostate epithelial cells.

Gossypol is an antisteroidogenic compound naturally found in cottonseed. Gossypol has been shown to inhibit steroidogenesis in the canine prostate and may inhibit canine prostate growth. Its mechanism of action, however, is largely unknown. Our laboratory has previously demonstrated that in vivo administration of gossypol to male dogs can reduce circulating levels of testosterone and estradiol. Gossypol also showed an ability to reduce prostate weights. To search for genes regulated by gossypol in the canine prostate, differential display RT-PCR was performed on total RNAs isolated from control and gossypol-treated male dogs. Gossypol was demonstrated to induce expression of spermidine/spermine-N1-acetyl-transferase (SSAT), the major catabolic enzyme for polyamines. This induction was confirmed by Northern hybridization analysis of total RNA isolated from prostates of mature dogs treated with gossypol for 2 months. Gossypol was also shown to inhibit the progression of cells into the S phase mediated by spermidine. Our findings support the notion that gossypol can inhibit prostate cell proliferation and may be a potential therapeutic agent for use in controlling overgrowth of the prostate.

Differential effect of keratinocyte growth factor (KGF) on aromatase activity in cultured canine prostatic epithelial cells.

We have recently cloned the mRNA encoding KGF from canine prostate and produced recombinant canine KGF (rcKGF) which specifically acts on cultured canine prostatic epithelial cells (CCPECs) which possess KGF receptors (Canatan et al., 1996; DNA Cell Biol. 15:247). In the present study, the effect of rcKGF on aromatase activity in CCPECs from young (6-month-old) and mature (3-year-old) dogs was examined. Release of 3H2O from labeled substrate was used as the indicator of aromatase activity. CCPECs were pulsed with [1-beta-3H]-androstenedione (1 microCi/ml, 6 hr). The amounts of 3H2O released into culture medium were measured (dpm) and total cellular proteins were determined. Aromatase activity was expressed as 3H2O dpm/mg cellular protein (mean +/- SEM). The basal level of aromatase activity in CCPECs from mature dogs was approximately 4 times higher (p < 0.05) than that in cells from young dogs. Aromatase activity in CCPECs from mature dogs increased in a dose-dependent manner upon treatment with rcKGF. Interestingly, rcKGF, at any of the concentrations tested, had no significant effect on aromatase activity in CCPECs from young dogs. These results are the first to indicate that aromatase activity is affected by KGF in mature CCPECs, suggesting that KGF may be involved indirectly in the etiology of benign prostatic hyperplasia by increasing aromatase activity and thus increasing aromatization of androgens. Aromatase induction by KGF may explain, at least in part, the increased aromatization of androgens observed in aged dogs. The exact mechanism of how KGF induces aromatase activity in CCPECs is needed to be addressed further.

Inhibition of human prostate cancer cells growth by gossypol is associated with stimulation of transforming growth factor-beta.

Gossypol (GP), an antifertility agent in males, is also capable of inhibiting the proliferation of a wide range of cancer cells in vivo and in vitro. Thus, in this study we investigated the effect of GP on the growth of human androgen-independent prostate cancer cell line (PC3). The results showed that GP acts as a potent inhibitor of PC3 cells as determined by thymidine incorporation assay and flow cytometric analysis. Flow cytometry revealed that treatment of PC3 cells with GP resulted in a dose- and time-dependent accumulation of cells in the GO/GI phase with a concomitant decrease in cells progressing to the S and G2/M phase. These data support our thymidine incorporation results which indicated that GP is a potent inhibitor of PC3 cells. By ribonuclease protection assay, we also investigated the effect of GP on transforming growth factor-beta 1 (TGF-beta 1) gene expression in PC3 cells. Interestingly, the stimulatory effect of GP on TGF-beta 1 gene expression correlates well with its inhibitory effect on PC3 cell DNA synthesis and its ability to arrest cells in GO/G1 phase. Based on these data, it can be concluded that GP is a potent inhibitor of prostate cancer cell growth that acts by arresting cells in GO/G1 phase and that this inhibitory effect may be mediated by TGF-beta 1.

Experimentally-induced prostatic hyperplasia in young beagles: a model to evaluate the chemotherapeutic effects of gossypol.

This study investigated the effect of gossypol on hyperplastic canine prostates induced with long-term administration of androgen and estrogen. Twelve 16-week-old male beagle dogs were divided evenly (n = 3) into 4 treatment groups: (1) CONTROL: vehicle only; (2) Gossypol-Treated: 20 mg/kg gossypol acetic acid; (3) Steroid-Induced: 4 mg/kg testosterone and 40 micrograms/kg estradiol-17 beta; (4) Gossypol-Treated and Steroid-Induced: 4 mg/kg testosterone, 40 micrograms/kg estradiol-17 beta and 20 mg/kg gossypol. The subjects received treatments every other day for 1 month. The beagles treated with steroids developed an acute enlargement (approximately 10-fold) of the prostate as compared to control. The prostatic acini were underdeveloped and characterized by simple squamous to low cuboidal epithelium in the control subjects while acini in steroid-induced subjects were characterized by simple tall columnar epithelium. The subjects treated with gossypol had prostates histologically similar to controls with the exception of loosened periurethral connective tissue. Serum testosterone and estradiol-17 beta levels imply that gossypol can reduce steroid hormonal levels. Mean serum testosterone levels in gossypol-treated subjects were reduced approximately 50% from controls. Serum biochemistry profiles indicate that steroid and/or gossypol treatments were not toxic at the doses and duration used in this study. These observations imply that gossypol and steroid hormones can interact to alter prostate development and gossypol metabolism and/or clearance.

Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF.

cDNA encoding the canine keratinocyte growth factor (KGF) was cloned from normal canine prostate tissue. The authentic canine KGF cDNA sequence, 686 bp in length, spans the protein-coding region and 88 bp of the 5' and 19 bp of the 3' untranslated regions of canine KGF. The predicted amino acid sequence of canine KGF is composed of 194 amino acid residues. Canine KGF exhibits highest homology with the human KGF cDNA and amino acid sequences (95.8% and 97.4%, respectively), while it demonstrates the lowest homology with the rat sequences at 88.0% and 92.3%, respectively. The degrees of homology with mouse cDNA and amino acid sequences are 91.8% and 95.9%, respectively. By using RNase protection assay, KGF was shown to be expressed in normal prostate tissues of both mature and young (5-month-old) dogs. In vitro, the recombinant canine KGF has mitogenic activity on cultured canine epithelial cells, whereas it has no effect on cultured canine prostatic stromal cells. This novel canine KGF cDNA may be a valuable tool in the study of human benign prostatic hyperplasia using the canine prostatic as a model.

Detection of keratinocyte growth factor (KGF) messenger ribonucleic acid and immunolocalization of KGF in the canine testis.

Keratinocyte growth factor (KGF) was originally discovered in human embryonic lung fibroblasts and is a member of the fibroblast growth factor (FGF) family. Members of the FGF family have been shown to regulate testicular function. However, the recently discovered KGF has not been studied in the testis. KGF has been detected in many other tissues, including the prostate, an organ whose development and function have been associated with presence of the testis. In this study, KGF mRNA was detected in the whole testis using reverse transcription polymerase chain reaction (RT-PCR). The 575-bp KGF-specific product was detected along with a 594-bp ß-actin-specific product. To identify the cell types in which KGF mRNA was predominantly expressed, interstitial cells were physically separated from seminiferous tubules. The interstitial cells were then sorted on a discontinuous Percoll gradient and total cellular mRNAs isolated. Using RT-PCR and Southern hybridization with specific cDNA probes, the KGF mRNA was detected in interstitial cells. KGF expression levels were then evaluated semiquantitatively with a competitive RT-PCR assay. KGF expression levels were highest in interstitial cells that equilibrated between 20 and 30% Percoll. Enriched Leydig cells and seminiferous tubules expressed low levels of KGF. Finally, immunohistochemical analysis was performed on canine testes using a rabbit anti-KGF polyclonal antibody. The KGF protein was localized predominantly to peritubular cells of the canine testis. These results suggest that KGF is synthesized in the canine testis.

Transforming growth factor-beta(1) regulates differentiation of porcine granulosa cells in vitro.

Transforming growth factor-beta (TGF-beta) is a potential regulator of ovarian function and follicular development. It is speculated that TGF-beta mediates the events in the follicle which culminate in ovulation of the oocyte. The complex processes which ultimately leads to this natural phenomenon must involve interactions between the 2 major follicular cell types, theca and granulosa cells, and the oocyte. Furthermore, a complex local regulatory system must exist to determine which follicles should undergo development and, eventually, which of those should ovulate or undergo atresia. To begin to understand this perplexing process, we must first understand the variables which control the function of each individual cell type. This study investigated the effect of TGF-beta(1) on FSH-induced porcine granulosa cell differentiation in vitro. Transforming growth factor-beta(1) was shown to inhibit progesterone production at high concentrations (0.1 and 10.0 ng/ml) after 12-, 24- and 48-hour treatment. However, TGF-beta(1) produced a biphasic effect on FSH-induced progesterone production during the 12-hour interval between the 36- and 48- hour treatment periods; TGF-beta(1) stimulated progesterone production at a low concentration (0.001 ng/ml) and inhibited production at high concentrations (0.1 and 10.0 ng/ml). The results obtained from the biphasic effect were not observed during any of the other incubation periods or intervals investigated. These results show that TGF-beta(1) has opposing effects on the differentiation of porcine granulosa cells as compared with those on rat granulosa cells. Moreover, TGF-beta(1) can produce opposing effects within the porcine granulosa cell itself which are specific to the concentration and treatment period used. The results of this study seem to suggest that TGF-beta(1) is species- and time-specific in its regulatory actions on FSH-induced porcine granulosa cell differentiation.

In vitro uptake and autoradiographic localization of tritiated gossypol in Taenia taeniaeformis metacestodes.

Gossypol, a natural polyphenolic compound, induces growth-inhibitory and antiparasitic effects in Taenia taeniaeformis metacestodes in vivo and in vitro. We investigated the uptake and localization of [3H]-gossypol in this parasite. Metacestodes were incubated in 10(-5) M [3H]-gossypol at 37 degrees C. Parasites steadily took up tritium activity over the first 3 h of incubation, after which a plateau was maintained for the duration of the experiment. Tissue: medium radioactivity ratios revealed that intralarval tritium activity matched extralarval activity within 30 min of incubation and continued to increase with time. Reverse-phase high-performance liquid chromatographic (HPLC) analysis confirmed tissue incorporation of tritium activity that manifested as a single radioactive species. Autoradiography localized [3H]-gossypol to the tegument, calcareous corpuscles, and parenchyma over the first 2 h of incubation. By 6 h, parenchymal radioactivity had disappeared. T. taeniaeformis metacestodes rapidly take up and accumulate [3H]-gossypol in vitro. This accumulation is apparently selective for specific sites, which may have implications for gossypol's metacestocidal action.