Antiangiogenic therapy and surgical practice.

BACKGROUND: Antiangiogenic therapy has become a reality with the recent introduction of bevacizumab, a monoclonal antibody against vascular endothelial growth factor. METHODS: Relevant medical literature from PubMed, National Institute for Health and Clinical Excellence and National Institutes of Health websites to August 2007 was reviewed. RESULTS AND CONCLUSIONS: Although often described as the fourth modality of treatment after surgery, radiotherapy and chemotherapy, many antiangiogenic drugs have failed to live up to expectations. Nevertheless, research continues and there are reasons to believe that antiangiogenic therapy may yet have a future in the clinical setting.

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Impaired detoxication of hydrogen sulfide in ulcerative colitis?

Impaired butyrate oxidation and raised counts of sulfate-reducing bacteria in the colon of patients with ulcerative colitis (UC) indicate that the disease may be induced or aggravated by hydrogen sulfide toxicity. We aimed to examine enzymatic removal of H(2)S in erythrocytes and colonic mucosa from controls and patients with UC and Crohn's disease (CD). Rhodanese (RHOD) and thiol methyltransferase (TMT) activities were measured in rectal mucosa and erythrocytes, and plasma thiocyanate was determined. Four groups were analyzed: patients with UC, patients with CD, hospital controls (patients with dyspepsia or IBS), and a group of healthy volunteers. RHOD and TMT activity in rectal biopsies did not differ significantly between controls and patients with UC or CD (n=56). Control levels of RHOD were significantly higher in men than in women (212+/-25 and 132+/-14 nmol/mg/min, respectively; P<0.01). In erythrocytes (n=128) RHOD activity was significantly higher in UC patients than in hospital or volunteer controls (1.15+/-0.12 compared with 0.88+/-0.12 and 0.66+/-0.02 nmol/mg/min; P<0.05 and P<0.02, respectively). TMT activity was also significantly higher in erythrocytes from UC patients and hospital controls than volunteer controls (2.02+/-0.13 pmol/mg/min [P<0.001], 1.51+/-0.21 pmol/mg/min [P<0.05], and 1.17+/-0.18 pmol/mg/min, respectively). We found no evidence of defective enzymic detoxication of sulfide by RHOD or TMT in patients with UC or CD.

Pituitary tumor transforming gene (PTTG) stimulates thyroid cell proliferation via a vascular endothelial growth factor/kinase insert domain receptor/inhibitor of DNA binding-3 autocrine pathway.

CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.

Sulfide-detoxifying enzymes in the human colon are decreased in cancer and upregulated in differentiation.

H2S is highly toxic and selectively inhibits butyrate oxidation in colonocytes. Ineffective detoxification may result in mucosal insult, inflammation, and ultimately in colorectal cancer (CRC). Rhodanese can detoxify H2S and is comprised of two isoenzymes: thiosulfate sulfurtransferase (TST) and mercaptopyruvate sulfurtransferase (MST). Using specific antisera to discriminate TST from MST, we found that only TST could detoxify H2S. In sections of normal colon, both enzymes were located on the luminal mucosal surface, and they were expressed in the colonocytes but not in the mucin-secreting goblet cells. Expression of both enzymes was focally lost in ulcerative colitis and markedly reduced in advanced colon cancer, the disease progression correlating with the decreased expression of MST and TST. In HT-29 cells, a human colon cancer cell line, TST activity and expression were significantly increased by butyrate and by histone deacetylase inhibition, agents that promote HT-29 cell differentiation. Sulfide (0.1 mM) also increased TST activity, but higher sulfide concentrations (0.3-3 mM) were toxic. Preincubation in butyrate to increase TST expression, decreased sensitivity of the cells to sulfide toxicity. We conclude that decreased expression of TST (or MST) is a tumor marker for CRC. TST expression is increased in colonocyte differentiation. Dysregulation of TST expression and activity resulting in inability to effectively detoxify could be a factor in the cell loss and inflammation that accompany ulcerative colitis and ultimately then in CRC.

Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer.

CONTEXT: Pituitary tumor-transforming gene (PTTG) is a multifunctional protein involved in several tumorigenic mechanisms, including angiogenesis. PTTG has been shown to promote angiogenesis, a key rate-limiting step in tumor progression, by up-regulation of fibroblast growth factor-2 and vascular endothelial growth factor. OBJECTIVE: To investigate whether PTTG regulates other angiogenic genes in thyroid cells, we performed angiogenesis-specific cDNA arrays after PTTG transfection. Two of the genes [inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1)] which showed differential expression in primary thyroid cells were validated in vitro and in vivo. RESULTS: TSP-1 showed a 2.5-fold reduction and ID3 showed a 3.5-fold induction in expression in response to PTTG overexpression in vitro. Conversely, suppression of PTTG with small interfering RNA was associated with a 2-fold induction of TSP-1 and a 2.2-fold reduction in ID3 expression. When we examined TSP-1 and ID3 expression in 34 differentiated thyroid cancers, ID3 was significantly increased in tumors compared with normal thyroid tissue. Furthermore, ID3 expression was significantly higher in follicular thyroid tumors than in papillary tumors. Although mean TSP-1 expression was not altered in cancers compared with normal thyroids, we observed a significant independent association between TSP-1 expression and early tumor recurrence, with recurrent tumors demonstrating 4.2-fold lower TSP-1 expression than normal thyroid tissues. CONCLUSION: We have identified ID3 and TSP-1 as two new downstream targets of PTTG in thyroid cancer. We propose that PTTG may promote angiogenesis by regulating the expression of multiple genes with both pro- and antiangiogenic properties and may thus be a key gene in triggering the angiogenic switch in thyroid tumorigenesis.

Endostatin expression in a pancreatic cell line is modulated by a TNFalpha-dependent elastase.

Endostatin, an inhibitor of angiogenesis, is a 20 kDa fragment of the basement membrane protein, collagen XVIII. The formation of endostatin relies upon the action of proteases on collagen XVIII. TNFalpha, produced by activated macrophages, is a multifunctional proinflammatory cytokine with known effects on endothelial function. We postulated that TNFalpha may modulate the activities of proteases and thus regulate endostatin formation in pancreatic cells. Collagen XVIII/endostatin mRNA was expressed in one pancreatic cell line, SUIT-2, but not in BxPc-3. The 20 kDa endostatin was found in the cell-conditioned medium of SUIT-2 cells. Precursor forms only were found in the cells. Exogenous endostatin was degraded by cellular lysates of SUIT-2 cells. Elastase activity was found in cell extracts but not the cell-conditioned media of SUIT-2 cells. Incubation of SUIT-2 cells with TNFalpha increased intracellular elastase activity and also increased secretion of endostatin into the medium. We conclude that endostatin is released by SUIT-2 cells and that increases in intracellular elastase, induced by TNFalpha, are correlated with increased secretion. Endostatin is however susceptible to degradation by intracellular proteases and if tissue injury accompanies inflammation, endostatin may be degraded, allowing angiogenesis to occur.

Endostatin expression in pancreatic tissue is modulated by elastase.

Pancreatic tumours are scirrhous, avascular tumours, suggesting that they may produce angiogenesis inhibitors that suppress the growth of the vasculature to the tumour and metastases. We have sought evidence for the angiogenesis inhibitor, endostatin, in normal and cancerous pancreatic tissue. Using Western blotting, we found mature 20 kDa endostatin in cancer tissue but not in normal tissue. Several endostatin-related peptides of higher mol wt were present in both tissues. Extracts from normal tissue were able to degrade exogenous endostatin, whereas extracts from cancer were without effect. Although the exocrine pancreas secretes inactive proenzymes of trypsin, chymotrypsin and elastase, their possible role in this degradation was examined. The trypsin/chymotrypsin inhibitor, Glycine max, did not prevent the degradation of endostatin by normal pancreatic extracts but elastatinal, a specific inhibitor of elastase, reduced the rate of degradation. Extracts of pancreatic tumours did not express any detectable elastase activity, but an elastase (Km 1.1 mM) was expressed by extracts of normal pancreas. We conclude that endostatin is present and stable in pancreatic cancer tissues, which may explain their avascular nature, but that normal pancreatic tissue expresses enzymes, including elastase, which rapidly degrade endostatin. The stability of endostatin may have implications for its therapeutic use.

PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro.

Human pituitary tumor-transforming gene (PTTG), known also as securin, is a multifunctional protein implicated in the control of mitosis and the pathogenesis of thyroid, colon, oesophageal and other tumour types. Critical to PTTG function is a C-terminal double PXXP motif, forming a putative SH3-interacting domain and housing the gene's sole reported phosphorylation site. The exact role of phosphorylation and PXXP structure in the modulation of PTTG action in vitro remains poorly understood. We therefore examined the mitotic, transformation, proliferation and transactivation function of the C-terminal PXXP motifs of human PTTG. Live-cell imaging studies using an EGFP-PTTG construct indicated that PTTG's regulation of mitosis is retained regardless of phosphorylation status. Colony-formation assays demonstrated that phosphorylation of PTTG may act as a potent inhibitor of cell transformation. In proliferation assays, NIH-3T3 cells stable transfected and overexpressing mutations preventing PTTG phosphorylation (Phos-) showed significantly increased [3H]thymidine incorporation compared with WT, whereas mutants mimicking constitutive phosphorylation of PTTG (Phos+) exhibited reduced cell proliferation. We demonstrated that PTTG transactivation of FGF-2 in primary thyroid and PTTG-null cell lines was not affected by PTTG phosphorylation but was prevented by a mutant disrupting the PXXP motifs (SH3-). Taken together, our data suggest that PXXP structure and phosphorylation are likely to exert independent and critical influences upon PTTG's diverse actions in vitro.

Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta.

Epidemiological studies of postmenopausal hormone replacement therapy show a reduction in the risk of developing colon cancer, and animal studies using 17beta-oestradiol (E(2)) demonstrate a decreased incidence of chemically-induced colon cancer. Using the colon cancer cell line, COLO205, we found that E(2) induced a dose-dependent increase in DNA fragmentation and nuclear condensation, significant effects being seen at 10(-12 )mol/l. BSA-conjugated E(2), which cannot enter cells, was ineffective at inducing apoptosis in COLO205 cells, indicating that E(2) was not acting through a cell-membrane receptor. E(2) did not induce the morphological changes characteristic of differentiation. Using RT-PCR we found that the oestrogen receptor alpha (ERalpha) isoform was absent in the COLO205 cell line in contrast to CACO-2, LoVo and SW620 cells, but mRNAs for ERbeta1, -beta2, -beta5 and -beta6 isoforms were detected. Western immunoblotting results showed full-length ERbeta protein but no detectable ERalpha in COLO205 cells. In normal human colon tissue samples immunoreactive ERbeta was found but ERalpha was barely detectable. Expression of ERbeta was lost in some colon cancer specimens and reduced in others. We conclude that E(2), through ERbeta, at concentrations found during replacement therapy, may inhibit the development of colon cancer by inducing apoptosis.

Thyroid follicular cells secrete plasminogen activators and can form angiostatin from plasminogen.

Angiostatin, a 38 kDa fragment of plasminogen, potently inhibits the growth of blood vessels. Angiostatin is generated from plasminogen by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in the presence of free sulphydryl donors. Angiogenesis inhibitors may be important in regulating angiogenesis in developing goitre. We have examined angiostatin formation in human primary thyrocyte cultures and a rat thyrocyte cell line (FRTL-5). We found that human thyroid cells in culture secrete plasminogen activators (both tPA and uPA) as well as matrix metalloproteinase 2 into the medium. When human thyrocyte conditioned medium was incubated with plasminogen (10 microg/ml) and N-acetylcysteine (100 microM) for 24 h, a 38 kDa fragment of plasminogen, which is consistent with angiostatin, was generated. The appearance of the 38 kDa fragment was increased by agents that increase cAMP (forskolin and 8 BrcAMP). FRTL-5 cells, which do not secrete uPA or tPA, did not generate angiostatin. Thyroid cells produce several angiogenic growth factors, and human thyrocyte conditioned medium stimulated growth of endothelial cells. When the conditioned medium was incubated with plasminogen and N-acetylcysteine, this stimulatory effect was lost, consistent with the production of a growth inhibitory factor. We conclude that thyroid cells can produce angiostatin from plasminogen in vitro, and this may play a role in vivo in limiting goitre size.

Sulphide-induced energy deficiency in colonic cells is prevented by glucose but not by butyrate.

BACKGROUND: In ulcerative colitis, hydrogen sulphide is postulated to impair colonocyte butyrate metabolism, leading to cellular energy deficiency and dysfunction. AIMS: To determine the effects of sulphide exposure on butyrate metabolism and adenosine triphosphate levels of HT29 colonic epithelial cancer cells, and to establish whether energy deficiency can be prevented by increased butyrate concentrations or the presence of glucose. METHODS: HT29 cells were maintained in medium containing 3 mM butyrate, 5 mM glucose, or both substrates. Oxidation rates were measured by 14CO2 release from 14C-labelled substrates. Cellular adenosine triphosphate was assayed using the luciferin/luciferase chemiluminescent method. The effects of sulphide (0-5 mM) on substrate oxidation and adenosine triphosphate levels and of increasing butyrate concentration (0-30 mM) with sulphide were observed. RESULTS: HT29 cells showed similar energy substrate usage to primary colonocyte cultures. Sulphide exposure inhibited butyrate oxidation and led to a reduction in cellular adenosine triphosphate. This fall was prevented by co-incubation with glucose, but not by increasing concentrations of butyrate. CONCLUSIONS: HT29 cells utilize butyrate as an energy substrate and represent a useful in vitro model of the effects of sulphide on colonocytes. Sulphide inhibits butyrate oxidation and leads to demonstrable energy deficiency, prevented by the presence of glucose but not by increased butyrate concentrations.

Mucosal protection against sulphide: importance of the enzyme rhodanese.

BACKGROUND: Hydrogen sulphide (H(2)S) is a potent toxin normally present in the colonic lumen which may play a role in ulcerative colitis (UC). Two enzymes, thiol methyltransferase (TMT) and rhodanese (RHOD), are thought to be responsible for sulphide removal but supportive evidence is lacking. AIMS: To determine the distribution of TMT and RHOD in different sites throughout the gastrointestinal tract and their efficacy as detoxifiers of H(2)S. METHODS: Enzyme activities were measured in normal tissue resected from patients with cancer. TMT and RHOD activities were determined using their conventional substrates, 2-mercaptoethanol and sodium thiosulphate, respectively. For measurement of H(2)S metabolism, sodium sulphide was used in the absence of dithiothreitol. Thiopurine methyltransferase (TPMT), which in common with TMT methylates sulphydryl groups but is not thought to act on H(2)S, was also examined. RESULTS: TMT, RHOD, and TPMT activities using their conventional substrates were found throughout the gastrointestinal tract with highest activity in the colonic mucosa. When H(2)S was given as substrate, no reaction product was found with TMT or TPMT but RHOD was extremely active (Km 8.8 mM, Vmax 14.6 nmol/mg/min). Incubation of colonic homogenates with a specific RHOD antibody prevented the metabolism of H(2)S, indicating that RHOD is responsible for detoxifying H(2)S. A purified preparation of RHOD also detoxified H(2)S. CONCLUSIONS: RHOD, located in the submucosa and crypts of the colon, is the principal enzyme involved in H(2)S detoxication. TMT does not participate in the detoxication of H(2)S.

Site-specific regulation of oestrogen receptor-alpha and -beta by oestradiol in human adipose tissue.

AIM: To examine the expression of oestrogen receptors alpha and beta (ERalpha and ERbeta) and their regulation by 17beta-oestradiol (E2) in stromal cells and adipocytes from human subcutaneous (s.c.) and omental (o.m.) adipose tissue. METHODS: Subcutaneous and o.m. abdominal adipose tissues were obtained from 10 women (mean age 63.5 +/- 4.8 years; mean weight 75.6 +/- 6.7 kg) undergoing elective or cosmetic surgery. Immunohistochemistry and RT-PCR analysis were used to detect the presence of ERalpha and ERbeta. The regulation of ERalpha and ERbeta by E(2) (10(-7) M to 10(-9) M) was examined using Western immunoblotting analysis in both s.c. and o.m. stromal cells and mature adipocytes cultured in serum-free, phenol red-free medium. RESULTS: Immunostaining of s.c. and o.m. adipose tissue showed that the ER subtypes were localized predominantly within the nucleus. Western analysis demonstrated that E2 treatments differentially altered ERalpha and ERbeta expression in s.c. and o.m. adipocytes. In s.c. and o.m. stromal cells, E(2) (10(-8) M) produced a significant up regulation relative to control of 66 kDa ERalpha (s.c.:1.87 +/- 0.22; o.m.:1.97 +/- 0.17; p < 0.05) and 60 kDa ERbeta (s.c.:1.66 +/- 0.3; o.m.: 1.68 +/- 0.16; p < 0.05). In s.c. adipocytes, however, ERalpha expression significantly decreased with E(2) 10(-8) M relative to control while ERbeta expression increased (ERalpha 0.58 +/- 0.06, ERbeta: 1.47 +/- 0.11; p < 0.05). In o.m. adipocytes, the inhibition of ERalpha with E(2) was not observed (ERalpha 1.86 +/- 0.36, ERbeta:1.03 +/- 0.15, p < 0.05) CONCLUSIONS: ERalpha and ERbeta are expressed but differentially regulated by E(2) in s.c. and o.m. adipocytes and stromal cells. The upregulation of ERbeta by E(2) suggests that E(2) maintains the expression of these receptors. The feed-back inhibition of ERalpha expression by E(2) in s.c. but not o.m. adipocytes observed in vitro is consistent with the data from ERalpha knock out mice where s.c. fat is increased. Selective ER modulators may have different effects in different adipose sites.

Dexamethasone inhibits tumor necrosis factor-alpha-induced apoptosis and interleukin-1 beta release in human subcutaneous adipocytes and preadipocytes.

Tumor necrosis factor-alpha (TNF alpha) can decrease adipose tissue mass, but in obesity, adipose tissue hypertrophy persists despite increased TNF alpha expression. The hormonal milieu of obesity may antagonize the adipostat effects of TNF alpha. We examined the effects of insulin and the synthetic glucocorticoid, dexamethasone (Dex), on TNF alpha-induced apoptosis and gene expression in human adipocytes and preadipocytes. Using RT multiplex PCR, the expression of the proapoptotic genes interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) and TNF alpha and the antiapoptotic genes bcl-2, nuclear factor-kappa B (NF kappa B), and NF kappa B inhibitory subunit, I kappa B, were examined. The expression and release of IL-1 beta, a postulated downstream effector of ICE-mediated apoptosis, were also determined. TNF alpha increased the messenger ribonucleic acid levels of ICE, TNF alpha, IL-1 beta, bcl-2, and NF kappa B in preadipocytes and adipocytes (P < 0.01). Dex inhibited TNFalpha-induced messenger ribonucleic acid expression of ICE, TNF alpha, and IL-1 beta (P < 0.01), but not that of bcl-2 and NF kappa B. TNF alpha stimulated IL-1 beta release from preadipocytes and adipocytes up to 20-fold, but the effect was abrogated by Dex. Apoptosis induced by TNF alpha was reduced to control levels (P < 0.01) by Dex. Insulin had no significant effect on TNF alpha-induced apoptosis and gene expression. In obesity, glucocorticoids may reduce TNF alpha actions in adipose tissue by inhibiting TNF alpha-induced apoptosis, IL-1 beta release, and TNF alpha expression.

Tie-2 is expressed on thyroid follicular cells, is increased in goiter, and is regulated by thyrotropin through cyclic adenosine 3',5'-monophosphate.

Angiogenesis is coordinated with follicular cell growth in goitrogenesis. The angiopoietins, Ang-1 and Ang-2, are angiogenic growth factors acting through Tie-2, a tyrosine kinase receptor. We have examined the expression and regulation of the angiopoietins and Tie-2 in human and rat thyroids. In human goiters there was increased Tie-2 immunostaining, compared with that in normal thyroids, on both follicular and endothelial cells. In an induced goiter in rats, in situ hybridization showed increased expression of messenger ribonucleic acids (mRNAs) for Tie-2 and Ang-1 in follicular cells. As Tie-2 has previously been believed to be restricted to cells of endothelial lineage in adults, we examined its expression further in isolated follicular cells. Tie-2 and Ang-1 mRNA expression in human thyrocytes was confirmed by ribonuclease protection assay. Ang-2 mRNA was not detected in human cultures or rat thyroids. In both human follicular cell cultures and FRTL-5 cells, immunoblotting showed that Tie-2 expression was increased by TSH and agents that increased intracellular cAMP. In conclusion, we have demonstrated the expression of Tie-2 and Ang-1 in thyroid epithelial and endothelial cells, and have shown the regulation of Tie-2 by TSH and cAMP in follicular cells. Tie-2 expression is increased in goiter in both humans and rats, consistent with a role in goitrogenesis.

Gender differences in the regulation of P450 aromatase expression and activity in human adipose tissue.

OBJECTIVE: To investigate the hormonal regulation of P450 aromatase activity (responsible for the conversion of C19 androgens to C18 oestrogens) in human adipose tissue from men and pre- and post-menopausal women. SUBJECTS: Subcutaneous abdominal adipose tissue was obtained from 19 subjects: six pre-menopausal females (mean age 41.8+/-(s.e.m.) 2.5; mean weight 76.01+/-5.6 kg), eight post-menopausal females (mean age 59.9+/-2.0; mean weight 63.5+/-2.6 kg), and five males (mean age 35.8+/-8.8; mean weight 78.5+/-7.8 kg) undergoing elective or cosmetic surgery. MEASUREMENTS: Cell viability and cell size were determined using staining techniques. RT-PCR was used to confirm the presence of aromatase. The regulation of aromatase activity was characterized using androstenedione as a substrate in a tritiated water release assay. Aromatase activity was analysed in abdominal subcutaneous stromal cells (ASC) and mature adipocytes (AD) cultured in serum-free medium with cortisol (10-6-10-7 M), insulin (500 nM) or a combination of both. RESULTS: In ASC aromatase activity increased in females from 14.5+/-1.7 to 29. 3+/-2.6 pmol/mg/h (n=14, P<0.05) and to 25.2+/-2.1 pmol/mg/h with cortisol (10-7 M) and insulin, respectively (P<0.05). In males ASC basal aromatase activity (20.5+/-4.2 pmol/mg/h; n=5) was inhibited by cortisol (10-7 M) alone (12.3+/-1.8 pmol/mg/h) and in combination with insulin (6.6+/-1.2 pmol/mg/h; men vs women, P<0.005). Aromatase activity in mature adipocytes was stimulated by cortisol plus insulin (P<0.05) with no gender-specific differences. Treatment of ASC from both pre- and post-menopausal females with cortisol alone (10-6 M; 10-7 M) or in combination with insulin demonstrated significantly different aromatase regulation compared with male aromatase stromal cell regulation (P<0.05); however there were no differences in aromatase regulation between pre- and post-menopausal females either in stromal cells or adipocytes. CONCLUSION: This study shows intrinsic gender differences in the regulation of aromatase, suggesting that differential enzyme regulation may affect sex steroid metabolism to alter the pattern of fat distribution between the sexes.

Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line.

BACKGROUND: Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). MATERIALS AND METHODS: Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, N(G)-monomethyl-L-arginine (L-NMMA) and Ro31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCalpha in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. RESULTS: PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by N(G)-monomethyl-L-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCalpha, betaII and iota were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCalpha and PKCiota within the time frame of NO release. The translocation of PKCalpha, but not PKCiota, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCalpha is the principal isozyme involved in this process and that eNOS may be a substrate for PKCalpha. Kinase assays performed using immunoprecipitated PKCalpha showed that PAF (1 nM) activated PKCalpha that was inhibited by co-incubation with Ro31-8220 and Ca(2+)-free medium. CONCLUSIONS: This study demonstrates that PAF-stimulated NO release via PKCalpha in epithelial cells might regulate endometrial functions such as implantation and menstruation.

Overexpression of protein kinase C-beta1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells.

BACKGROUND & AIMS: We have previously reported that nonsteroidal anti-inflammatory drugs (NSAIDs) could induce apoptosis of gastric epithelial cells both in vivo and in vitro. This study investigated the role of protein kinase C (PKC) isoforms in the regulation of NSAID-induced apoptosis. METHODS: Protein levels of 12 PKC isoforms in AGS cells, in the presence or absence of indomethacin, were determined by Western blot. The effect of PKC-beta1 overexpression by transfection with its complementary DNA (cDNA) on indomethacin-induced apoptosis and apoptosis-related genes, including p53, p21(waf1/cip1), and c-myc, was further investigated. RESULTS: Treatment with indomethacin decreased the abundance of PKC-beta1 and increased that of PKC-beta2, eta, and epsilon, but did not alter the expression of PKC alpha, gamma, zeta, delta, iota, and micro. Overexpression of PKC-beta1 attenuated the apoptotic response of AGS cells to indomethacin, associated with overexpression of p21(waf1/cip1) in both messenger RNA and protein levels. Inhibition of PKC-beta1-mediated overexpression of p21(waf1/cip1) by its antisense cDNA partially reduced the antiapoptotic effect of PKC-beta1. CONCLUSIONS: Indomethacin-induced apoptosis in gastric cancer cells is partly mediated by differential regulation of PKC isoform expression. Enhanced expression of exogenous PKC-beta1 protects against indomethacin-induced apoptosis through up-regulation of p21(waf1/cip1).

Tumour necrosis factor-alpha exerts dual effects on human adipose leptin synthesis and release.

The acute and chronic effects of tumour necrosis factor-alpha (TNF-alpha) on leptin production by human preadipocytes, differentiated preadipocytes, and mature adipocytes have been examined by competitive RT-PCR of leptin mRNA and by western blotting. In preadipocytes, secreted leptin was detectable after 5-day incubation in differentiation medium and this increased 4-fold by day 20. TNF-alpha blocked leptin synthesis during differentiation. In differentiated preadipocytes and mature adipocytes, TNF-alpha treatment resulted in time-dependent decreases in mRNA for leptin and glycerol-3-phosphate dehydrogenase (G3PD). In contrast, TNF-alpha (4-8-h treatment) resulted in a 4-fold increase in leptin release. This effect was lost at 24 h and leptin accumulation in culture medium was decreased 24-48 h after TNF-alpha addition. We conclude that TNF-alpha stimulates the release of preformed leptin from human mature adipocytes and existing differentiated preadipocytes, which may contribute to obesity/infection-linked hyperleptinemia, and that TNF-alpha inhibits leptin synthesis via inhibition of preadipocyte differentiation and induction of adipocyte dedifferentiation.