Anti-cancer actions of a recombinant antibody (R6313/G2) against the angiotensin II AT1 receptor.

Although several tumour types express both AT1 and AT2 angiotensin II receptors, and angiotensin II stimulates cell proliferation, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are not effective anti-cancer agents. Development of a biologically active monoclonal antibody (6313/G2) against the AT1 receptor prompted the testing of a recombinant short-chain variable fragment form (R6313/G2) against breast cancer cells in vitro and in vivo. Cell lines MCF-7, MDA-MB-231 and T47D all expressed both receptor subtypes. In vitro, R6313/G2 suppressed cell proliferation in the presence of 100 nM angiotensin II, with IC50s of 30 nM, 153 nM and 2.8 microM for the three cell types respectively; in contrast, the AT1 receptor blocker losartan was effective only in T47D cells, at 25 microM. Studies on MCF-7 and T47D cells showed R6313/G2 also opposed the angiotensin II-induced inhibition of caspase-3/7 activity. In vivo, hollow fibres containing the cell lines were implanted in nu/nu balb-c mice at two sites, s.c. and i.p. Treatments of R6313/G2 at 2.5 nmol/kg and 25 nmol/kg twice per day for 7 days dose dependently reduced cell numbers for all three cell lines, but here MCF-7 cells responded most sensitively and MDA-MB-231 cells least. Although T47D cells were refractory at the s.c. site, growth was inhibited at the i.p. location, and otherwise results were similar at the two sites. In xenografts, MCF-7 cell tumours were dose dependently reduced by R6313/G2, and 13 and 27 nmol/kg R6313/G2 twice/day gave means of 74 and 76% tumour regression after 7 days. The data suggest that the anti-cancer action of R6313/G2 is considerably more effective than AT1 antagonists.

The role of angiotensin II in the regulation of breast cancer cell adhesion and invasion.

As breast cancer remains the most common cause of cancer death in women, there is a continuing need not only to further characterise the processes of cancer progression, but also to improve accuracy of prognostic markers. Breast epithelial cells express components of the renin angiotensin system and studies suggest that these may be altered in disease progression. In addition, altered integrin expression correlates with lymph node metastasis. The aim of this study was to investigate the relationship between angiotensin II (AII) and integrins in breast tissue and, in particular, their role in breast cancer cell metastasis. Using in vitro assays, AII (10(-6) M)-treated MCF-7 and T47D breast cancer cells both show reduced adhesion to extracellular matrix proteins collagen-, fibronectin- and laminin-coated wells (P<0.001) and reduced invasion through collagen-, fibronectin- and laminin-coated membranes (P<0.05). This action was inhibited by co-treatment with the angiotensin type 1 receptor (AT1R) antagonist losartan (10(-5) M). The addition of the AT2R inhibitor PD123319 (10(-5) M) to AII-treated cells had no significant effect. Semi-quantitative reverse transcriptase-PCR and western blotting revealed that cells treated with AII (10(-6) M) expressed lower levels of both integrin alpha3 and beta1. Using specific inhibitors, this was shown to occur through protein kinase C signalling. These data suggest that AII reduces cell adhesion and invasion through the type 1 receptor and that this effect may be due to reduced expression of integrins, and in particular alpha3 and beta1.

Regulation of hepatic steroid receptors and enzymes by the 3beta-hydroxysteroid dehydrogenase inhibitor trilostane.

Therapies designed to treat hypercortisolism have usually sought to reduce circulating glucocorticoid concentrations, however the local tissue endocrine environment could be an alternative target. The 3beta-hydroxysteroid dehydrogenase Delta5-4 isomerase (3beta-HSD) inhibitor trilostane is of interest, since, although it is only moderately and transiently effective in reducing circulating steroid, it is remarkably effective in alleviating Cushing's symptoms in veterinary applications. To seek alternative modes of action, male Wistar rats were treated with trilostane. Although final circulating corticosteroid concentrations were unaffected, liver 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) transcription and translation was significantly increased, whereas 3beta-HSD was not affected either in liver or adrenal. Glucocorticoid receptor (GR) mRNA was down-regulated, and mineralocorticoid receptor (MR) up-regulated by trilostane treatment: no changes in 11beta-HSD1 mRNA were observed. Trilostane also had no direct effect on GR response element-mediated gene transcription. The results show that the tissue endocrine environment is affected by trilostane treatment in the absence of sustained changes in circulating corticosteroid. The combination of increased 11beta-HSD2 and reduced GR expression in target organs could be expected to ameliorate the effects of excess glucocorticoid, suggesting new therapeutic approaches.

Localisation of renin-angiotensin system (RAS) components in breast.

Angiotensin II has mitogenic and angiogenic effects and its receptors are widespread, particularly in epithelial tissue. Tissue renin angiotensin systems (tRASs) may be a local source of angiotensin II that has specific paracrine functions. To investigate the presence of a tRAS in normal human breast and tumours. Immunocytochemistry, and quantitative RT-PCR was used to establish: (i) the presence and localisation of RAS components, (ii) the possibility of their involvement in cancer. (1) mRNA coding for angiotensinogen, prorenin, angiotensin converting enzyme (ACE), and both AT1 and AT2 receptors was demonstrated in normal and diseased breast tissues. (2) (pro)renin was identified in epithelial cells in both normal and diseased tissue, but in invasive carcinoma, its distribution was mostly confined to fibroblasts or could not be detected at all. (3) Angiotensin converting enzyme was shown in epithelial cells in both normal and malignant tissue. The results are consistent with the hypothesis that a tRAS is present in the breast, and is disrupted in invasive cancer.

Distribution of extracellular signal-regulated protein kinases 1 and 2 in the rat adrenal and their activation by angiotensin II.

The adrenal gland of the rat is continuously regenerated through proliferation of a stem cell population in the outer part of the gland. To clarify the location of proliferative events within the adrenal gland, and the factors that stimulate them, rat adrenal capsule preparations, consisting of capsule, zona glomerulosa (ZG) and the outer zona fasciculata (ZF) were maintained in vitro under different conditions of stimulation, for varying periods. Sites of proliferation were identified by 5-bromo-2'-deoxy-uridine (BrdU) staining, and the distribution of classical MAP kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) 1 and 2, immunoreactivity was determined using immunocytochemistry. BrdU staining was limited to the outer glomerulosa and the capsule, where it was enhanced by angiotensin II, but not by a high potassium ion concentration nor by ACTH. In contrast, ERK1/2 immunoreactivity was distributed throughout the ZG and in the medulla, with none detectable in the ZF and reticularis. Furthermore, angiotensin II, potassium ions and ACTH were all shown to induce ERK1 and ERK2 phosphorylation in the ZG. Treatment of adrenal capsule tissue with the specific MAPK kinase inhibitor PD98059 revealed inhibition of ERK1/2 phosphorylation, but no effect on angiotensin II-induced aldosterone secretion. Although the distribution and activation of the MAPK pathway suggest a link with proliferation, the findings clearly designated only the outer part of the glomerulosa and capsule as a potential stem cell population. Further functions should be sought for the apparently silent major part of the glomerulosa.

Manganese superoxide dismutase activity in the rat adrenal.

In the light of studies suggesting that transcription of the gene coding for manganese superoxide dismutase (MnSOD) is induced by ACTH in the rat adrenal gland, northern blot analysis was used to determine its mRNA distribution. It was found that mRNA coding for MnSOD is primarily present in the inner zones of the rat adrenal cortex, and not the glomerulosa. To investigate the functional relationships between MnSOD activity and expression and adrenocortical function, adrenals and blood were taken from animals pretreated with corticotrophin or betamethasone (Betnesol), or subjected to a low-sodium diet. MnSOD activity in inner zone mitochondrial fractions was enhanced by corticotrophin and by a low-sodium diet, but suppressed by betamethasone. Apparent cytosolic MnSOD activity, total cytosolic MnSOD and CuZnMn-SOD, and glomerulosa mitochondrial MnSOD all were unaffected. Steroid assays showed a clear correlation between circulating corticosterone and inner zone mitochondrial MnSOD, but none between aldosterone and glomerulosa MnSOD. Immunoblot analysis of MnSOD showed two apparent isoforms, at approximately 25 kDa and 75 kDa. There was a partial relationship between expression of the 75 kDa isoform and MnSOD activity, in that it was induced by corticotrophin. However, there was also a slight induction with betamethasone, and a low-sodium diet had no effect. The 25 kDa MnSOD isoform was unaffected by the treatments. The results suggest that MnSOD may have a specific role in the steroidogenic function of the fasciculata/reticularis of the rat adrenal, but not in that of the glomerulosa.

Glomerulosa function and aldosterone synthesis in the rat.

Since its discovery, it has been generally assumed that the primary function of the zona glomerulosa of the adrenal cortex is the secretion of aldosterone. Taking evidence from the rat, and recognising that there is probably considerable species variation, I argue here that the glomerulosa in fact has many functions, including aldosterone synthesis, but is probably only a relatively poor de novo source of steroid. In vitro, the CYP11B2 (aldosterone synthase) of the glomerulosa can and does utilise as substrates products arising from CYP11B1 (11beta-hydroxylase) activity in fasciculata cells. Whether it does in vivo is open to question, but corticosterone and 18-hydroxydeoxycorticosterone are both present in circulating rat plasma at suitable concentrations. Such a mechanism would explain several inconsistencies in the literature, including the anomalous distribution of steroidogenic enzymes in the glomerulosa, the stimulation of CYP11B1 products by aldosterone secretagogues such as potassium ions or angiotensin II, the partial dependence of aldosterone secretion in vivo on an intact pituitary, the sensitivity of aldosterone secretion to tissue disruption in vitro, and the "late pathway" regulation of aldosterone synthesis.

ACTH modulates ERK phosphorylation in the adrenal gland in a time-dependent manner.

ACTH is known to act through the activation of cAMP/PKA in adrenocortical cells, but it has been suggested that it could also act via other pathways such as the ERK 1/2 cascade. To determine the effects of ACTH administration at sequential time points on the activation of ERKs 1/2, groups of rats (n = 6/group) were subjected to i.p. injections of either ACTH (Synacthen Depot-0.2 mg/Kg), or saline (Ct). The animals were sacrificed and the adrenal glands collected at different timings after ACTH injection (2 h, 18 h and 24 h). Two additional groups were injected daily until sacrifice (3 days and 15 days). Blood was collected for analysis and the adrenals were used for immunohistochemistry or Western Blot (WB) analysis. Immunoreactivity was scored by counting the mean number of zonae fasciculata (ZF) and reticularis (ZR) positive cells/section (mean +/- SEM). Adrenal weight was increased by ACTH in comparison with Ct. Corticosterone levels, as expected, were higher in ACTH treated animals than in Ct. The number of pERK positive cells increased in a time-dependent manner until 3d, and declined although not significantly in the 15 days animals (Control--48.13 +/- 9.0; ACTH 2 h--125.93 +/- 14.5; ACTH 18 h-139.46 +/- 10.0; ACTH 24 h--185.28 +/- 13.3; ACTH 3 days--198.47 +/- 18.6; ACTH 15 days--158.58 +/- 15.1). Comparable results were obtained with WB analysis. Our data shows that ACTH induces the activation of the MAPK/ERKs 1/2 cascade, especially in the ZF, consistent with this zone being more responsive to ACTH.

Effects of injury and progesterone treatment on progesterone receptor and progesterone binding protein 25-Dx expression in the rat spinal cord.

Progesterone provides neuroprotection after spinal cord injury, but the molecular mechanisms involved in this effect are not completely understood. In this work, expression of two binding proteins for progesterone was studied in intact and injured rat spinal cord: the classical intracellular progesterone receptor (PR) and 25-Dx, a recently discovered progesterone membrane binding site. RT-PCR was employed to determine their relative mRNA levels, whereas cellular localization and relative protein levels were investigated by immunocytochemistry. We observed that spinal cord PR mRNA was not up-regulated by estrogen in contrast to what is observed in many brain areas and in the uterus, but was abundant as it amounted to a third of that measured in the estradiol-stimulated uterus. In male rats with complete spinal cord transection, levels of PR mRNA were significantly decreased, while those of 25-Dx mRNA remained unchanged with respect to control animals. When spinal cord-injured animals received progesterone treatment during 72 h, PR mRNA levels were not affected and remained low, whereas 25-Dx mRNA levels were significantly increased. Immunostaining of PR showed its intracellular localization in both neurons and glial cells, whereas 25-Dx immunoreactivity was localized to cell membranes of dorsal horn and central canal neurons. As the two binding proteins for progesterone differ with respect to their response to lesion, their regulation by progesterone, their cellular and subcellular localizations, their functions may differ under normal and pathological conditions. These observations point to a novel and potentially important role of the progesterone binding protein 25-Dx after injury of the nervous system and suggest that the neuroprotective effects of progesterone may not necessarily be mediated by the classical progesterone receptor but may involve distinct membrane binding sites.

Adrenocortical zonation and ACTH.

The clear morphological distinction between the cells of the different adrenocortical zones has attracted speculation and experiment to interpret their functions and the ways in which they are regulated. Considerable data have been produced in recent years that has benefited a fuller understanding of the processes of steroidogenesis and of cell proliferation at the molecular level. This now enables the reexamination of earlier concepts. It is evident that there is considerable species variation, and this article, dealing mainly with the rat, reaches conclusions that do not necessarily apply to other mammals. In the rat adrenal, however, the evidence suggests that the greatest differences between the functions of the zones are between the glomerulosa and the fasciculata. Here the sometimes all-or-nothing demarcation in their complement of components associated with steroidogenesis or with cell proliferation suggests a stark division of labor. In this model the fasciculata is the main engine of steroid hormone output and the glomerulosa is the site of cell proliferation, recruitment, and differentiation. Regulating these functions are angiotensin II and other paracrine components that modulate and maintain the glomerulosa, and ACTH, that maintains the fasciculata, and recruits new fasciculata cells by transformation of proliferating glomerulosa cells. Grafted onto this mostly vegetative function of the glomerulosa is CYP11B2, limited to just a fraction of the outer glomerulosa in rats on a normal laboratory diet and generating aldosterone (and 18-hydroxycorticosterone) from precursors whose origin is not, from the evidence summarized here, very clear, but may include the fasciculata, directly or indirectly. The biosynthesis of aldosterone in the rat certainly requires reinterpretation.

Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro.

Angiotensin II type 1 receptors have been identified in Fallopian tube epithelia. Polarized confluent human Fallopian tube epithelial cell cultures were used under short-circuit conditions to study the actions of angiotensin II on electrogenic ion transport. The results demonstrate that angiotensin II increases baseline short-circuit current, implying a net transport of negatively charged ions from a basal to apical direction. This effect was inhibited by the selective angiotensin II type 1 receptor antagonist losartan. The effects of angiotensin II on short-circuit current were rapid in onset, brief in duration, and although less than those achieved with ATP, similar in amplitude to those described for other epithelia with angiotensin II. These findings reflect a significant retention of function for these cells in monolayer culture. Immunohistochemistry using the antibody 6313/G2, which is directed against a specific sequence in the extracellular domain of the angiotensin II type 1 receptor, confirmed that the receptor was retained in cultured cells. The results indicate that angiotensin II plays a role in regulating the composition of Fallopian tube secretions.

Neuropeptides and adrenocortical proliferation in vitro.

Vasoactive intestinal peptide (VIP) and Neuropeptide Y (NPY) are localised in the capsule and zona glomerulosa region of the adrenal cortex, where they play an important role in regulating steroidogenesis and adrenal blood flow. This study investigates the effect of these neuropeptides on adrenocortical cellular proliferation and steroidogenesis in vitro. Capsular/glomerulosa and innerzone/medulla preparations were either stimulated acutely with NPY or VIP (both 10(-6) M) for up to 2 hours or for 24 hours, four and eight days in vitro in eagles MEM (3.4 mM K+). DNA synthesis was determined using immunocytochemistry through the incorporation of the thymidine analogue 5-bromo-2'-deoxyridine (BrdU, 20 mg/mL). Phosphorylation of mitogen activated protein kinase ERK1/2 was assessed by western blotting. Both VIP (10(-6) M) and NPY (10(-6) M) treatment caused an increase in DNA synthesis after four days in culture. Acute NPY treatment caused an increase in ERK1 and 2 phosphorylation (p < 0.01) in the capsular/zona glomerulosa. Vasoactive intestinal peptide treatment caused a significant increase in ERK 1/2 phosphorylation (p < 0.05) only in innerzones/medulla preparations. Both responses were maximal between 10 and 30 min of incubation and decrease thereafter. These data provide further evidence for the role of the mitogen activated protein kinases ERK1 and 2 in the proliferative events in the adrenal gland and demonstrate stimulation of cell division by the adrenal neuropeptides VIP and NPY in vitro.

Proliferation of capsular stem cells induced by ACTH in the rat adrenal cortex.

Despite great efforts devoted to clarifying the localization of proliferative activity in the adrenal cortex, the agents that stimulate proliferation remain controversial, and the nature of the stem cells from which cortical cells differentiate is incompletely understood. We studied proliferative activity in the rat adrenal cortex using an immunohistochemical method to detect the presence of the Proliferating Cell Nuclear Antigen (PCNA) (an intranuclear enzyme whose synthesis reaches the maximum intensity during the S-phase of the cell cycle). Groups of six rats were subjected to daily intraperitoneal injection of either corticotropin (ACTH1-24--0.2 mg/kg), dexamethasone (Dexa--4 mg/kg) or 0.9% saline for three consecutive days and killed 24 h after the last injection. Adrenal weight was significantly increased by ACTH treatment and reduced by Dexa. Concentrations of endogenous ACTH in plasma were lower in the Dexa group than in controls, and curiously, this was true in the ACTH1-24 treated group as well, probably in consequence of the increased corticosterone levels providing negative feedback at the hypothalamic-pituitary level. Corticosterone levels, as expected, were increased by the ACTH stimulus and reduced by the use of Dexa. Proliferating cell nuclear antigen immunostaining was close to zero in Dexa treated animals and low in controls. In ACTH treated rats, a significantly increased number of cells were positively stained. Positive cells were identified in both in zona glomerulosa (ZG) and zona intermedia (ZI) but many were located in the capsule. Zona fasciculata (ZF) and zona reticularis (ZR) were devoid of staining in all of these cases. We conclude that pharmacological doses of ACTH induce proliferation of capsular fibroblasts. Following descriptions by early 20th century researchers it is possible that these cells may also be stem cells and differentiate into adrenal cortex cells.

The effect of cAMP on ion transport in Fallopian tube epithelial cells in vitro.

The coupled movement of ions and water across epithelia determines the composition and volume of fluid present in the lumen of organs. The second messenger cAMP is important in effecting electrolyte and water transport in many transporting epithelia; however, its role in Fallopian tube transport is uncertain. We have conducted electrophysiological studies on Fallopian tube epithelial cell monolayers in Ussing chambers and have demonstrated that exogenously added cAMP and agents that generate its intracellular production results in an increase in short-circuit current consistent with the transport of net electrical charge from a basal to mucosal direction. In contrast to the known effects of ATP in this tissue, the increase in short-circuit current was not explicable in terms of electrogenic chloride secretion as it was not affected by the chloride channel inhibitors, 4-acetamido-4'-isothiocyanostilbene-2,2-disulphonic acid 1 mmol/l (SITS) and frusemide. Instead the current was reduced by the sodium channel inhibitor, amiloride, and was therefore, in part, explicable in terms of electrogenic Na+ absorption. These findings will enhance our understanding of the physiological mechanisms responsible for human Fallopian tubal fluid formation and composition.

Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function.

Fertilization-promoting peptide (FPP) regulates the adenylyl cyclase (AC)/cAMP pathway to elicit capacitation-dependent responses, stimulating capacitation in uncapacitated spermatozoa and then arresting it in capacitated cells, thereby inhibiting spontaneous acrosome reactions. Like FPP, calcitonin and angiotensin II are found in seminal plasma and so might affect sperm function; this study investigated responses in uncapacitated and capacitated mouse spermatozoa to these three peptides. Both calcitonin (5 ng/ml) and angiotensin II (1 and 10nmol/l), like FPP (100nmol/l), significantly stimulated capacitation, assessed using chlortetracycline (CTC) fluorescence and fertilization in vitro analyses. Combinations of two or three peptides, at high and low, non-stimulatory concentrations, were more stimulatory than the individual peptides, suggesting that they may act on the same signalling pathway, plausibly AC/cAMP; preliminary data indicate that calcitonin does stimulate cAMP production. In capacitated cells, FPP and calcitonin elicited pertussis toxin-sensitive inhibition of spontaneous acrosome loss, suggesting involvement of inhibitory G proteins; angiotensin II had no detectable effect. When all three peptides were used, angiotensin II did not interfere with inhibitory responses to FPP/calcitonin. These results suggest that angiotensin II, calcitonin and FPP may somehow modulate the AC/cAMP signal transduction pathway, but the precise mechanisms involved have yet to be elucidated.

Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor.

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.

Angiotensin II in human seminal fluid.

The renin-angiotensin system (RAS) and angiotensin II are important in sperm function and male fertility. Angiotensin II type I (AT1) receptors have been identified in developing and ejaculated human spermatozoa, and angiotensin can stimulate sperm motility, the acrosome reaction and binding to the zona pellucida. However, there is little information on the availability of the hormone to spermatozoa during the reproductive process. Seminal plasma and blood plasma obtained from normal and subfertile subjects was extracted, and angiotensin content was analysed by radioimmunoassay. Values obtained for blood angiotensin II were within the normal range at 16.0 +/- 3.1 pg/ml (mean +/- SEM). Values for seminal plasma were usually 3-5 fold higher, at 51.6 +/- 9.3 pg/ml (n = 34, P < 0.0001). High performance liquid chromatography analysis showed that approximately 80% of the immunoreactive angiotensin was attributable to angiotensin II itself. However, seminal plasma angiotensin II concentrations were not correlated with blood angiotensin II, sperm concentration or sperm motility. The results show that immunoreactive angiotensin from a source other than the circulation is available to spermatozoa in human ejaculates. The results are consistent with the concept that angiotensin II has an important role in male fertility.

Aldosterone mediates angiotensin II-stimulated rat vascular smooth muscle cell proliferation.

Aldosterone, possibly locally generated, has been suggested to have a role in potentiating angiotensin II (AII)-stimulated hypertrophy of cultured vascular smooth muscle cells. To examine the possibility that aldosterone may mediate the proliferative actions of AII, rat aortic smooth muscle cells (RASMCs) in culture were treated with AII in the presence and absence of the specific AII type 1 receptor (AT1) antagonist, losartan, and aldosterone was assayed in culture medium extracts by radioimmunoassay. AII significantly enhanced aldosterone formation (at 10(-8) M: 123.8+/-14.85 vs control 71. 28+/- 8.71 fmol/10(5) cells, P<0.05; at 10(-7) M: 172.38+/-33.44, P<0.05), but not in the presence of losartan (at 10(-8) M: 53. 71+/-18.73, P>0.05; at 10(-7) M: 89.68+/-25.05, P>0.05). In other studies, the reverse transcriptase-polymerase chain reaction, performed on RNA extracted from RASMCs using aldosterone synthase (CYP11B2) specific primers, gave a single band of about 268 bp, consistent with that expected for the enzyme. Finally, using [(3)H]methylthymidine uptake as an index of cellular proliferation, tritium incorporation was increased in the AII-treated group at concentrations greater than 10(-10) M. The aldosterone antagonist, spironolactone (10(-5) M), inhibited the incorporation of [(3)H]thymidine into RASMCs stimulated by AII. These results suggest that locally generated aldosterone may mediate the effects of AII, acting via the AT1 receptor, in stimulating RASMC proliferation.

Integrin beta1 upregulation in MCF-7 breast cancer cells by angiotensin II.

AIMS: Integrins are a major family of cell adhesion molecules whose function is perturbed in tumour invasion and metastasis. Angiotensin II (A II) is well-known in the systemic control of water and electrolyte homeostasis and haemodynamics, but recent evidence points to an additional local renin-angiotensin system (RAS) with possible long-term trophic effects including carcinogenesis. METHODS: The effect of angiotensin II on MCF-7 human breast cancer cell line integrin expression was evaluated with immunocytochemistry (ICC) and immunoprecipitation (IP). RESULTS: The experiments demonstrated a 1.40 +/- 0.14-fold increase in beta, integrin expression on MCF-7 cells following treatment with A II. CONCLUSIONS: These findings report the first evidence of an association between integrins and the RAS in human breast cancer cells and suggest a novel research avenue for future anti-metastatic strategies, through the manipulation of cell adhesion mechanics, in the management of invasive human breast cancer.

The expression of renin and the formation of angiotensin II in bovine aortic endothelial cells.

One controversy in the field of vascular angiotensin generation has surrounded the nature and particularly the source of vascular renin. This study investigated the expression of renin protein and its mRNA in aortic endothelial cells using immunocytochemistry, Western blotting, in situ hybridization and reverse transcription PCR (RT-PCR). Using a monoclonal antibody against human renin, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Immunoblotting of solubilized proteins separated by SDS-PAGE from cultured aortic endothelial cells identified two immunoreactive species with molecular masses of approximately 37-40 kDa. In situ hybridization showed that renin mRNA was localized in the cytoplasm of these cells. Using RT-PCR of RNA extracted from bovine aortic endothelial cells with primers specific for human renin, a clear single band was detected, which had the predicted size of 142 bp for (pro)renin. Angiotensin II (Ang II) was assayed in conditioned medium (CM) from cultured bovine aortic endothelial cells, and in addition, the effects of Ang II and CM on the proliferation of aorta smooth muscle cells (ASMC) were also studied. The results showed that CM contained Ang II equivalent to 15.05+/-4.67 pg/10(6) cells. Assay of smooth muscle cell proliferation by cell number, and by tritiated thymidine uptake, showed that proliferative responses in the presence of Ang II at a concentration of 10(-6)M were evident within 1 day of subculture, and cell numbers were nearly twice those of controls after 2 days. Thymidine incorporation into ASMC was also increased by Ang II in a dose-dependent manner and by endothelial cell CM. In both cases, stimulated proliferation was inhibited by the Ang II type 1 (AT1) receptor selective antagonist, losartan. These findings suggest that these vascular endothelial cells are a source of locally synthesized renin that may thus be involved in vascular Ang II generation. They also suggest that Ang II produced by the endothelial cells may be secreted and stimulate ASMC proliferation via the AT1 receptor.