Human endometrium as a neuroendocrine tissue: expression, regulation and biological roles of endometrial corticotropin-releasing hormone (CRH) and opioid peptides.
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Animals↗
Biomedical subjects
C Stournaras
Publications and source records attributed to C Stournaras.
Estrogens and glucocorticoids induce the expression of c-erbB2/NEU receptor in Ishikawa human endometrial cells.
We investigated the effects of estrogens and other steroid hormones on c-erbB2 gene expression in Ishikawa human endometrial adenocarcinoma cells. We have found that the c-erbB2/NEU transcripts are present in the Ishikawa endometrial cell line as well as in human endometrial adenocarcinoma cells. Both cell types express the 4.6 and 2.3 kb c-erbB2 mRNAs. Estradiol significantly increased in a time- and dose-dependent manner the content of c-erbB2 mRNA and the concentration of NEU protein in Ishikawa cell extracts, while progesterone was devoid of any activity. The effect of estradiol was partially reversed by the antiestrogen 4-hydroxytamoxifen, which, however, given alone exhibited agonist effects. Glucocorticoid dexamethasone augmented in a time- and dose-dependent fashion the content of c-erbB2 mRNA and the concentration of NEU protein in Ishikawa cell extracts. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist increasing c-erbB2 gene activation. To our knowledge, this is the first report documenting the induction by steroid hormones of c-erbB2 gene expression in neoplastic human endometrial cells. Our data support the hypothesis that the oncogenic effect of estrogens on human endometrial cells may be partially mediated by its effect on the expression of the c-erbB2 proto-oncogene. The finding that glucocorticoids may induce endometrial c-erbB2 gene expression suggests that they may participate in the emergence of uterine neoplasias.
Adenocarcinoma↗
Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth.
Opioids and somatostatin analogs have been implicated in the modulation of renal water handling, but whether their action is accomplished through central and/or peripheral mechanisms remains controversial. In different cell systems, on the other hand, opioids and somatostatin inhibit cell proliferation. In the present study, we have used an established cell line, derived from opossum kidney (OK) proximal tubules, in order to characterize opioid and somatostatin receptors and to investigate the action of opioids and somatostatin on tubular epithelial tissue. Our results show the presence of one class of opioid binding sites with kappa, selectivity (KD 4.6 +/- 0.9 nM, 57,250 sites/cell), whereas delta, mu, or other subtypes of the kappa site were absent. Somatostatin presents also a high affinity site on these cells (KD 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the NA+/Pi cotransporter was observed, indicating that these agents do not affect ion transport mechanisms. However, opioid agonists and somatostatin analogs decrease OK cell proliferation in a dose-dependent manner; in the same nanomolar concentration range, they displayed reversible specific binding for these agents. The addition of diprenorphine, a general opioid antagonist, reversed the effects of opioids, with the exception of morphine. Furthermore, morphine interacts with the somatostatin receptor in this cell line too, as was the case in the breast cancer T47D cell line. Our results indicate that in the proximal tubule opioids and somatostatin do not affect transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair.
Animals↗
Altered actin polymerization dynamics in various malignant cell types: evidence for differential sensitivity to cytochalasin B.
Using the DNase I inhibition assay, fluorimetric measurements, and immunoblot analysis, we studied quantitatively changes in the actin polymerization dynamics in primary cultures of normal and malignant human lymphocytes, normal human endometrial cells, and in various leukemic and endometrial adenocarcinoma cell lines. The G/total-actin ratio of malignant cells was found to be 1.37 to 1.81-fold higher compared to normal cells, indicating that malignant cells express reduced amounts of polymerized actin. The above findings were corroborated by fluorescence measurements of the amounts of rhodamine-phalloidin-labeled F-actin in normal and neoplastic cells, which showed significantly lower F-actin content in malignant cell preparations. Moreover, the total actin content, as quantitated by the DNase I inhibition assay and by immunoblot analysis, was found to be significantly decreased in the primary cultures of malignant human lymphocytes and endometrial cells when compared to the total actin levels in corresponding normal cells. Proliferation and viability measurements of normal and neoplastic cells in culture, treated equally with cytochalasin B (CB), revealed an increased susceptibility of malignant cells to this anticytoskeletal agent. This was not due to increased CB incorporation in neoplastic cells, as indicated by 3H-CB uptake experiments. In addition, fluorescence microscopy, in the presence of graded concentrations of CB, showed destabilization of microfilaments in the poorly differentiated endometrial adenocarcinoma HEC-50 cells, compared to the well-differentiated Ishikawa cells. In conclusion, all investigated malignant cells are characterized by: (a) higher G/total-actin ratio; (b) decreased F- and total-actin content; and (c) lower resistance to CB treatment. These quantitatively determined parameters may represent potential biochemical indicators reflecting malignant transformation. Moreover, it seems worthwhile to explore whether or not the differential sensitivity of malignant cells to anticytoskeletal drugs may provide a valuable approach to the manipulation of malignant cells.
Actins↗
Na+/Pi co-transport alters rapidly cytoskeletal protein polymerization dynamics in opossum kidney cells.
We studied with biochemical and immunofluorescent techniques the interactions between the actin microfilament and tubulin microtubule cytoskeleton and Na+/P1 co-transport in opossum kidney cells, a line with proximal tubular characteristics. On brief (5 min) incubation of the cells with a low (0.1 mM) concentration of Pi, a rapid F-actin depolymerization takes place, which fails to occur in cells incubated under similar conditions with 1 mM Pi. The disassembly of actin microfilaments could be quantitatively expressed as a 33% increase in the ration of monomeric G-actin to polymerized F-actin (G/F-actin ration from 0.80 +/- 0.03 to 1.06 +/- 0.06, n = 28, P<0.01), owing to a significant decrease in the latter. Under these conditions microfilaments were also markedly destabilized, as shown by their diminished resistance to graded cytochalasin B concentrations. In addition, incubation of opossum kidney cells with low Pi concentrations (0.1 mM) resulted within 5 min in a substantial depolymerization of microtubules, shown by immunofluorescence microscopy and measured as a 70.9 +/- 6.9% (n = 11, P<0.01) decrement by immunoblot analysis. These changes, which occur only when extracellular Pi concentrations are kept low, seem to be related to a significant increase within 5 min in the rate of cellular Pi uptake by 25.5% under these conditions. The shifts in the dynamic equilibria between monomeric and polymerized actin and tubulin in response to cellular Pi uptake were transient, being fully reversible within 30 min. Moreover, the effect of Pi seemed to be specific because inhibition of its uptake by phosphonoformic acid blunted microtubular disassembly markedly. In contrast, measurement of Pi uptake in the presence of agents known to stabilize cytoskeletal structures showed a substantial decrease with phallacidin, which stabilized microfilaments, whereas the microtubule stabilizer taxol had no apparent effect. These results indicate that acute alterations in the polymerization dynamics and stability of both microfilaments and microtubules are involved in the modulation of Na+/Pi co-transport and suggest important cytoskeletal participation in proximal tubular transport functions.
Actin Cytoskeleton↗
Corticotropin releasing hormone (CRH) in normal and pregnant uterus: physiological implications.
Corticotropin-releasing hormone, is a hypothalamic neuropeptide, responsible not only for the endocrine but also the autonomic, immunological and behavioural responses of mammalian organisms to stress. CRH is also expressed in female reproductive tissues, such as placenta and uterus. Multiple sites within the pregnant uterine cavity express the CRH gene, including the trophoblasts, fetal membranes (chorion, amnion) and decidua. The trophoblastic syncytium appears to be the major source of placental CRH. It is postulated that placental CRH influences the HPA axis of either mother or fetus and participates at the initiation of labour. Recent findings show that human and rat uterus express the CRH gene. Epithelial cells of both species are the main source of endometrial CRH, while stroma does not seem to express it, unless it differentiates to decidua. Estrogens and glucocorticoids inhibit and prostaglandin E2 stimulates the promoter of human CRH gene in transfected human endometrial cells, suggesting that endometrial CRH gene expression is under the control of these agents. Moreover, in rats, endometrial CRH expression is significantly higher at the implantation sites, compared to that at the inter-implantation uterine regions. Given the proinflammatory/vasoregulatory properties of CRH, we hypothesize that endometrial CRH may participate in the regulation of intrauterine phenomena, such as blastocyst implantation, endometrial vascularization and myometrial contractility.
Animals↗
Dexamethasone alters rapidly actin polymerization dynamics in human endometrial cells: evidence for nongenomic actions involving cAMP turnover.
Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total-actin ratio in a time- and dose-dependent manner. Specifically, in untreated Ishikawa cells the G/total-actin ratio was 0.48 +/- 0.01 (n = 26). It became 0.35 +/- 0.01 (n = 13, P < 0.01) following exposure to 10(-7) M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G-actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F-actin content by 44% (n = 6, P < 0.001) in cells treated with dexamethasone (10(-7)M, 15 min). The rapid dexamethasone-induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10(-7)M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non-transcriptional pathways, since actinomycin D did not block the dexamethasone-induced actin polymerization. In addition, cell treatment with 10(-7)M dexamethasone for 15 min fully reversed the forskolin-, but not the 8-bromo-cAMP-induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10(-7)M dexamethasone (n = 4, P < 0.01). In conclusion, our results showed that dexamethasone induces rapid, time-, and dose-dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids.
Actin Cytoskeleton↗
Regulation of the promoter of the human corticotropin-releasing hormone gene in transfected human endometrial cells.
Corticotropin-releasing hormone (CRH) is expressed in several peripheral tissues, including normal epithelial cells of the human and rodent uterus. However, the biological role of endometrial CRH is known in neither species. As a first step to clarify this role, we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase. Transfected cells were exposed for 18 h to 8-bromo cyclic adenosine monophosphate, forskolin, epidermal growth factor, steroids (estradiol, progesterone, and the synthetic glucocorticoid dexamethasone and their antagonists), and prostaglandin E2; then the activity of the luciferase reporter was determined in the cell lysates. We found that the activity of the 5'-flanking region of the CRH gene was stimulated by cyclic adenosine monophosphate and epidermal growth factor and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist, suppressing the CRH gene activation, while progesterone was devoid of any activity. Prostaglandin E2 stimulated the CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of prostaglandin E2.
Adenocarcinoma↗
PC12 cells as a model to study the effects of opioids on normal and tumoral adrenal chromaffin cells.
Normal adrenal chromaffin cells produce delta opioid peptides while at the same time they have mainly kappa opioid receptors. This paper describes our date regarding the expression of the prodynorphin gene, the precursor of a family of endogenous kappa opioid ligands, in the PC12 rat pheochromocytoma cell line, and the effects of synthetic kappa opioid agonists and of Naloxone on various aspects of PC12 cell function including their secretion of catecholamines, proliferation and differentiation. This is the first part of a series of projects aimed at studying, (a) the conditions under which the prodynorphin gene is expressed in normal adrenomedullary cells, and (b) the physiological role of the endogenous kappa opioids in the physiology of the adrenal medulla.
Adrenal Medulla↗
Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus.
We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that: a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.
Animals↗
The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium.
CRH is produced by several intrauterine sites, including placenta and desidua, during pregnancy. However, no data are available regarding the presence of CRH in the nonpregnant uterus. We now report that CRH is produced in the epithelial cells of normally cycling human uterus and in an endometrial epithelial cell-derived tumor. Specifically, we have found that: 1) Northern blot hybridization analysis of normal glandular endometrium as well as of Ishikawa human endometrial adenocarcinoma cells showed the presence of the CRH messenger RNA; the size of the transcript seemed to be identical to that present in human placenta and rat hypothalamus; 2) immunoreactive CRH (ir-CRH) was detectable in normal dispersed glandular endometrial cells as well as in the Ishikawa adenocarcinoma cells; 3) gel filtration chromatography of normal glandular endometrial and Ishikawa cell extracts and their culture media showed that most ir-CRH present had the mol wt of the authentic CRH peptide; in addition, a larger form of ir-CRH was also present in both normal and tumoral endometrial epithelial cell extracts; the latter most probably correspondents to CRH precursor molecules; and 4) immunofluorescence staining of CRH in normal glandular endometrial and Ishikawa cells revealed a cytoplasm rich in granules positive for ir-CRH. Our findings suggest that CRH may play an important role in the physiological events taking place within the uterine cavity, since CRH seems to be present in nonpregnant as well as pregnant uteri. Since CRH is expressed in normal endometrial epithelial cells and in an epithelial tumoral cell line, we propose the use of the Ishikawa cell line as a convenient model for the in vitro study of endometrial CRH.
Adenocarcinoma↗
Interaction between steroid hormones and endometrial opioids.
The opioids beta-endorphin and the dynorphins belong to two separate families of endogenous opioid peptides (EOP). They are produced not only in the central nervous system but also in nonneural tissues where, as it appears, they act locally via paracrine mechanisms. These opioids have been shown to be produced at multiple sites along the mammalian reproductive tract including the intrauterine cavity. The aim of the present work was to find out if the well differentiated human endometrial cell line of Ishikawa, which has been shown to be a good in vitro model for the study of the effects of steroid hormones on human epithelial endometrium, expresses these two EOP. Northern blot hybridization of RNA from these cells showed the presence of a 1.2-kb POMC and a 2.4-kb PDYN transcript. Radioimmunoassay and gel filtration chromatography characterization of the immunoreactive (IR) opioid peptides present in the culture media showed the presence of IR-beta-endorphin and IR-dynorphins. The apparent molecular weight of IR-beta-endorphin was that of authentic beta-endorphin while the bulk of the IR-dynorphin had an apparent molecular weight of 8 kd. The secretion of both opioids could be increased by KCl-induced depolarization. Estrogen and glucocorticoids decreased, in a dose- and time-dependent manner, the secretion of beta-endorphin from the Ishikawa cells while progesterone and dihydrotestosterone did not have a statistically significant effect. The antiprogestin-antiglucocorticoid RU486 acted as an agonist, i.e., it diminished beta-endorphin secretion possibly via glucocorticoid receptors. On the other hand, the secretion of dynorphins was not affected by any of the steroids tested while LHRH, the inducer of gonadotropins and anterior pituitary dynorphins secretion, provoked a time- and dose-dependent increase of their secretion without affecting that of beta-endorphin. These data suggest that the regulation of endometrial opioids production is type-specific. Thus, it is possible that each type of endometrial opioid participates in different local homeostatic loops and exerts distinct paracrine effects.
Adenocarcinoma↗
Cytochalasin B may shorten actin filaments by a mechanism independent of barbed end capping.
It is generally accepted that cytochalasin B (CB), as well as other cytochalasins, shorten actin filaments by blocking monomer addition at the fast-growing ("barbed") end of these polymers. Despite the predominance of this mechanism, recent evidence suggests that other interactions may also occur between CB and F-actin. To investigate this possibility further we have employed an actin derivative, prepared by substitution at Cys374 by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several ionic conditions. We further show that in the presence of CB the glutathionyl-F-actin exhibits a significantly higher ATPase activity than the non-modified F-actin. These data argue that the incorporation of glutathionyl groups prevents the high-affinity binding of CB to the barbed end of actin filaments, probably due to a decreased hydrophobicity of the CB binding site by the introduction of the hydrophilic glutathionyl residue. Despite the lack of substantial binding at equilibrium, we have found that the addition of CB to glutathionyl-F-actin results in extensive fragmentation of the filaments, as demonstrated by electron microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from barbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cytochalasins and with the physiologically important actin capping/severing proteins.
Actins↗
Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes.
The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0.30 +/- 0.03 (mean +/- SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0.49 +/- 0.03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0.5 +/- 0.07 (n = 4), indicating a 1.7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10(-6) and 10(-5) M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10(-4) M), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted.(ABSTRACT TRUNCATED AT 250 WORDS)
Actin Cytoskeleton↗
In vitro differential effects of the antiglucocorticoid RU486 on the release of lymphokines from mitogen-activated normal human lymphocytes.
The synthetic antiglucocorticoid RU486 has multiple effects on the immune system. We have recently reported that RU486 suppresses normal lymphocyte proliferation and downregulates interleukin-2 receptors (IL-2R) by decreasing the accumulation of the beta-chain IL-2R mRNA in normal human lymphocytes in culture. To further explore the mechanism of the immunoregulatory actions of RU486, in the present study, we investigated the effects of this molecule on the release of lymphokines from phytohemagglutinin (PHA)-activated normal human peripheral blood lymphocytes (NPBL) in culture. We have found that RU486 differentially regulates the release of lymphokines from PHA-activated NPB lymphocytes. Specifically, RU486 (at concentrations of 1-100 nM) exerts pure antagonist actions by almost completely reversing the inhibitory effects of the glucocorticoid dexamethasone (Dex) on the release of monocyte/macrophages-derived lymphokines, such as IL-1, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha). Dex decreased in a dose-dependent manner the release of the above four lymphokines, with an ID50 of 0.9 +/- 0.1, 4.76 +/- 0.4, 9.8 +/- 1.8, and 1.16 +/- 0.2 nM for IL-1, IL-6, IL-8 and TNF-alpha, respectively. Conversely, RU486 exhibits both agonist and antagonist effects on the release of T-lymphocyte-derived lymphokines. RU486 given alone, exerts agonist/glucocorticoid effects, by decreasing in a dose-dependent manner the release of IL-2 and -3. The maximal inhibitory effect of RU486 was observed at 10 nM and was 64.5 +/- 4.3% of the control value, (n = 6, P < 0.02) for IL-2 and 59.2 +/- 6.3% (n = 6, P < 0.02) for IL-3. The ID50 of RU486 for the release of IL-2 and -3 were 14.6 +/- 2.0 and 11.6 +/- 1.9 nM, respectively, i.e. almost similar with those of Dex. Interestingly, when high doses of RU486 (1 microM) were combined with Dex RU486 exhibited antagonist actions by significantly counteracting the inhibitory effects of Dex on IL-2 and -3 release. In conclusion, the antiglucocorticoid RU486 exhibits complex regulatory actions on lymphokine secretion, dependent upon the type of the lymphokine-producing cell. A pure antagonist effect was observed on the release of monocyte-derived IL-1, IL-6, IL-8 and TNF-alpha. However, when RU486 was given alone it acted as a glucocorticoid agonist on the secretion of T-lymphocyte-derived IL-2 and -3, while combined with the agonist (Dex) it exhibits antagonist effects on the release of the above lymphokines.(ABSTRACT TRUNCATED AT 400 WORDS)
Cells, Cultured↗
Hepatocyte swelling leads to rapid decrease of the G-/total actin ratio and increases actin mRNA levels.
Exposure of isolated rat hepatocytes to hypotonic (190 mosmol/l) incubation media lowered the cellular G-actin level without affecting the total actin content: here the G-/total actin ratio decreased by 15.5 +/- 1.4% (n = 7). Similar effects were observed following isotonic cell swelling by either addition of glutamine (10 mM) or insulin (100 nM), resulting in a decrease of the G-/total actin ratios by 13.5 +/- 2.1% (n = 5) and 14.1 +/- 1.1% (n = 11), respectively. The effects of hypotonic exposure, glutamine and insulin on the G-/total actin ratio largely occurred within 1 min and persisted for at least 2 h in presence of the respective effectors. After a 120 min exposure to hypotonic media, glutamine or insulin the actin mRNA levels were increased 2.4-, 2.0- and 3.6-fold, respectively. Hypertonic exposure lowered the G-/total actin ratio by only 4.9 +/- 2.5% (n = 4) and increased actin mRNA levels only 1.2-fold. There was a close relationship between glutamine- and hypotonicity-induced cell swelling and the decrease of G-/total actin ratios. The data suggest that cell swelling exerts rapid and marked effects on the state of actin polymerization and increases actin mRNA levels. Thus, cytoskeletal alterations in response to cell swelling may be involved in the regulation of hepatic metabolism by cell volume.
Actins↗
Normal and Ha-ras-1 oncogene transformed Buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors.
In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha-ras-1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10(-5) M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1-1 cells were not seriously affected; a higher cytochalasin B concentration (> or = 2 x 10(-5) M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10(-6) M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cells, without affecting their counterparts in the transformed BRLHO6T1-1 cells. A 10-fold higher drug concentration (10(-5) M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]-cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1-1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha-ras-1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape.(ABSTRACT TRUNCATED AT 250 WORDS)
Actin Cytoskeleton↗
Heat shock protein HSP90 and its association with the cytoskeleton: a morphological study.
To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenocarcinoma↗