Induction by sphingomyelinase of shiga toxin receptor and shiga toxin 2 sensitivity in human microvascular endothelial cells.

Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells.

Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca(2+)-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1-5 mm) and medium-sized (5-8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells. Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-beta1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-beta1 dose-response studies revealed an ED(50) of 0. 24-0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion. Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca(2+)-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 microM) stimulated intracellular free calcium ion concentrations ([Ca(2+)](i)) in single porcine theca cells. The [Ca(2+)](i) elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca(2+)](i) response. Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-beta and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.

Expression and specific immunolocalization of the human parathyroid hormone/parathyroid hormone-related protein receptor in the uteroplacental unit.

OBJECTIVE: Our purpose in these studies was to determine the expression and cellular localization of the parathyroid hormone/parathyroid hormone-related protein receptor in the human uteroplacental unit. STUDY DESIGN: Human uteroplacental tissues were obtained and ribonucleic acid was extracted. Reverse transcriptase-polymerase chain reaction was performed with use of primers for both the parathyroid hormone/parathyroid hormone-related protein receptor and human phosphoglyceraldehyde dehydrogenase. Ethidium bromide-stained gels and Southern blots were evaluated, and polymerase chain reaction fragments were sequenced. For immunohistochemistry, slides were incubated with a newly developed antibody (3D1.1) specific for the parathyroid hormone/parathyroid hormone-related protein receptor, and bound monoclonal antibody was detected by use of the avidin-biotin technique. RESULTS: Reverse transcriptase polymerase chain reaction gels and blots showed that receptor messenger ribonucleic acid was present in choriodecidua, placenta, and myometrium. Sequence analysis revealed complete identity of the receptor product and the known nucleotide sequence in the receptor. There was intense receptor staining of the myometrial smooth muscle as well as staining of the endothelium and smooth muscle of the associated vasculature. In umbilical cord immunoreactive receptor was found in the vascular endothelium and vascular smooth muscle cells and in stromal cells. In choriodecidua receptor was found in chorionic trophoblasts and decidualized endometrial stromal cells. In all tissues immunostaining was specific, as evidenced by the blocking of staining after addition of receptor peptide to the antibody (absorbed controls). CONCLUSION: The parathyroid hormone/parathyroid hormone-related protein receptor is widely expressed in the human uteroplacental unit. The cellular localizations of the receptor in smooth muscle reflect the ability of parathyroid hormone-related protein to relax both uterine and vascular smooth muscle. The presence of novel autocrine and paracrine systems in the human uteroplacental unit is suggested by the finding that the same cells or adjacent cells produce both parathyroid hormone-related protein and its receptor.

Parathyroid hormone-related protein mRNA in sheep endometrium and myometrium during late gestation and labor.

OBJECTIVE: To determine quantitative changes of parathyroid hormone-related protein (PTHrP) mRNA in sheep myometrium and endometrium during late gestation and labor. METHODS: Tissues were obtained from 22 pregnant ewes under halothane anesthesia: early controls at 131 days' gestational age (dGA) not in labor (ECNL; n = 6); during cortisol-induced premature labor (CPL at 131 dGA; n = 6); in term spontaneous labor (STL at 140-145 dGA; n = 5); or term control animals not in labor (TCNL at 140-145 dGA; n = 5). Total RNA was extracted and subjected to Northern blot analysis. Blots were probed with a human PTHrP cDNA probe, stripped, and rehybridized with an 18s rRNA nucleotide probe to normalize PTHrP mRNA levels. RESULTS: Endometrial PTHrP mRNA:18s was unaffected by gestational age or labor in tissues from the four groups. In contrast, myometrial PTHrP mRNA:18s ratio was decreased when TCNL and STL were compared with both ECNL and CPL groups (P < .05). CONCLUSIONS: Significant changes occur at the end of gestation in pregnant ovine myometrium PTHrP mRNA but not in the endometrium. In myometrium PTHrP mRNA levels were down-regulated at term regardless of whether labor was present. It seems that PTHrP mRNA levels in myometrium are related to gestational age rather than to labor per se in sheep. We hypothesize that PTHrP may play a role in maintaining uterine quiescence until term, at which time levels fall allowing myometrial contractile activity to increase unopposed by PTHrP.

Interleukin-1 beta and interleukin-4 increase parathyroid hormone-related protein secretion by human umbilical vein endothelial cells in culture.

OBJECTIVE: Our purpose was to learn whether cytokines such as interleukin-1 beta and related (or antagonistic) cytokines, hormones, and growth factors could regulate secretion of the vasorelaxant parathyroid hormone-related protein in human umbilical vein endothelial cells in culture. STUDY DESIGN: Secondary cultures of human umbilical vein endothelial cells were grown to confluence and treated with interleukin-1 beta, an array of factors with possible regulatory actions (cytokines, growth factors, vasoactive peptides, and steroids), and a phorbol ester as a stimulatory control. After 24 hours immunoreactive parathyroid hormone-related peptide in the media was measured by a two-site sandwich radioimmunoassay. The mechanism of interleukin-1 beta action was probed with interleukin-1 beta receptor antagonist and selected inhibitors. RESULTS: Interleukin-1 beta (10 ng/ml) produced up to an eightfold increase in parathyroid hormone-related peptide secretion from human umbilical vein endothelial cells in culture (p < 0.01). Half-maximal stimulation was seen at 0.23 ng/ml. Interleukin-1 receptor antagonist, cycloheximide, and actinomycin D blocked the effects of interleukin-1 beta (p < 0.05). Interleukin-4 at 10 ng/ml and phorbol 12-myristate 13-acetate at 10(-7) mol/L significantly increased the secretion of parathyroid hormone-related peptide by human umbilical vein endothelial cells (p < 0.05). The time course of each interleukin showed an effect beyond 12 hours. The effects of interleukin-1 beta and interleukin-4 appeared to be specific, because a large series of related interleukins and other growth factors and cytokines were without effect. CONCLUSION: Interleukin-1 beta and interleukin-4 increase parathyroid hormone-related protein secretion in human umbilical vein endothelial cells in culture. Because interleukin-1 beta messenger ribonucleic acid has been found in umbilical cord endothelial cells, we propose that the umbilical cord has a novel vasorelaxant regulatory system that uses interleukin-1 beta endothelial action and secretion of the vasorelaxant parathyroid hormone-related peptide.

Expression of parathyroid hormone-related protein and its receptor in human umbilical cord: evidence for a paracrine system involving umbilical vessels.

OBJECTIVE: Our purpose was to study the expression and localization of parathyroid hormone-related protein and expression of the parathyroid hormone/parathyroid hormone-related protein receptor in human umbilical cord. STUDY DESIGN: The expression and localization of parathyroid hormone-related protein and expression of the parathyroid hormone/parathyroid hormone-related protein receptor were studied in isolated tissues from the human umbilical cord by Northern analysis, reverse-transcriptase polymerase chain reaction, Southern gel analysis, and immunolocalization procedures at the light and electron microscopic levels. RESULTS: Parathyroid hormone-related protein was abundantly expressed in the umbilical cord. Immunohistochemical and immunocytochemical techniques confirmed hormone localization in the amnion epithelial layer and in vascular smooth muscle and endothelial cells in vessels from the umbilical cord and placental chorionic plate. Multiplex reverse-transcriptase polymerase chain reaction identified expression of receptor messenger ribonucleic acid in vessels of the umbilical cord; this finding was verified by means of Southern gel analysis of the products of the reverse-transcriptase polymerase chain reaction. CONCLUSION: A parathyroid hormone-related protein paracrine system appears to exist in human umbilical cord. We suggest that it may be involved in the control of fetal placental circulation.

Sensitization to hyperthermia by 3,3'-dipentyloxacarbocyanine iodide: a positive correlation with DNA damage and negative correlations with altered cell morphology, oxygen consumption inhibition, and reduced ATP levels.

The cyanine dye 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) (concentrations of 0.5 microgram/ml to 5.0 micrograms/ml) was shown to be a potent sensitizer of Chinese hamster ovary (CHO) cells to hyperthermic cell killing at 43.0 degrees C or 45.5 degrees C, while exhibiting no cytotoxicity at 37.0 degrees C. Sensitization to hyperthermic cell killing was accompanied by an increase in damage to the DNA, as measured by DNA unwinding. The increased DNA damage correlated qualitatively with the enhanced heat killing induced by DiOC5(3). This correlation was better in cells heated at 43.0 degrees C than in those heated at 45.5 degrees C. DiOC5(3) is known to affect other cellular functions. It inhibits electron transport, uncouples oxidative phosphorylation, and inhibits calcium ATPases. The effects of DiOC5(3) on oxygen consumption and ATP content were therefore measured at 37.0 degrees C and at hyperthermic temperatures. The results demonstrated that inhibition of oxygen consumption and reduction of cellular ATP levels played no role in inducing heat sensitization in DiOC5(3)-treated cells, or in causing cell death in cells treated with heat alone.

A DNA double-strand break repair-deficient mutant of CHO cells shows reduced radiosensitization after exposure to hyperthermic temperatures in the plateau phase of growth.

Heat response and heat-induced radiosensitization were studied in plateau-phase cultures of CHO cells and their radiation-sensitive counterpart, the xrs-5 cells. The xrs-5 cells were more sensitive to heat alone than were CHO cells. A large enhancement in radiation-induced killing was observed in CHO cells pre-exposed to heat (43 degrees C), expressed as a reduction in the values of Do and Dq. Contrary to the results obtained with CHO cells, pre-exposure to heat of xrs-5 cells affected radiation sensitivity to a much lesser extent. D1, the radiation dose required to reduce cell survival to 1%, decreased in CHO cells from 8.7 Gy to 2.5 Gy with increasing heat damage (cell survival after exposure to heat alone), whereas it decreased from 1.4 Gy to 0.9 Gy in xrs-5 cells. These results suggest that heat-induced radiosensitization is compromised in plateau-phase xrs-5 cells. Since xrs-5 cells are deficient in DNA dsb repair, it is hypothesized that DNA dsb repair proficiency is a prerequisite for heat radiosensitization and that heat-induced inhibition of DNA dsb repair is likely to contribute to the radiosensitization observed in repair-proficient cell lines after exposure to high temperatures.

Effects of hyperthermia on the repair of radiation-induced DNA single- and double-strand breaks in DNA double-strand break repair-deficient and repair-proficient cell lines.

The effect of heat on the induction and repair of DNA single (ssb) and double (dsb) strand breaks was studied in irradiated exponentially growing or plateau-phase CHO cells and their DNA dsb repair-deficient, radiation-sensitive counterpart, the xrs-5 cells. Induction and repair of DNA ssb was measured by the alkaline unwinding technique, whereas induction and repair of DNA dsb was measured by the non-unwinding filter elution technique. The results indicated that pre-exposure of cells to heat (45 x 5 degrees C) for 8-30 min did not affect the induction of DNA ssb or DNA dsb per Gy and dalton of DNA in CHO or xrs-5 cells, tested either in the exponential or in the plateau-phase of growth. On the other hand, pre-exposure to heat inhibited DNA repair processes and increased the fraction of unrepaired radiation-induced damage measured 2 h after irradiation. Repair of DNA dsb was more heat-sensitive than repair of DNA ssb in both cell lines. Repair of radiation-induced ssb or dsb was inhibited in xrs-5 cells to a larger extent than in CHO cells after a similar exposure to heat. These results complement those previously reported on heat-induced radiosensitization in these cell lines, and suggest that the reduction in heat-induced radiosensitization observed in xrs-5 cells is largely due to their deficiency in repairing DNA dsb, rather than to a reduction in the ability of heat to inhibit DNA repair processes in general. The data presented here provide further support to the hypothesis that DNA dsb repair proficiency is a prerequisite for heat-induced radiosensitization.

Comparative studies on repair inhibition by araA, araC and aphidicolin of radiation induced DNA and chromosome damage in rodent cells: comparison with fixation of PLD.

The effect of araA and araC, two specific inhibitors of DNA polymerases alpha and beta, and of aphidicolin, a specific inhibitor of DNA polymerase alpha, on repair of radiation induced damage was studied at the DNA, the chromosome and the cell level in plateau-phase CHO cells. For the experiments at the chromosome level, the premature chromosome condensation technique was used. Repair of total DNA breaks was measured by the unwinding technique and repair of DNA double strand breaks by the neutral filter elution technique. In agreement with the hypothesis that DNA polymerase activity is involved in cellular repair reactions, araA and aphidicolin strongly inhibited repair of radiation induced damage at the DNA and the chromosome level. Also, araC inhibited repair at these endpoints but only to a very limited extent. The relative inhibition of repair by these compounds was similar at the various endpoints studied. At the survival level, araA effectively fixed radiation induced PLD resulting in survival levels corresponding to an exponential survival curve. On the other hand, araC and aphidicolin had only a small effect on cell survival. DNA replication was effectively inhibited by aphidicolin, moderately by araC, and even less by araA. These observations demonstrate that the efficacy of a polymerase inhibitor to inhibit DNA and chromosome repair does not always coincide with its ability to fix radiation induced PLD, or with its ability to inhibit DNA replication. This finding indicates that partly different biochemical pathways may underlie PLD fixation, DNA repair inhibition, and DNA replication inhibition.

Radiosensitive xrs-5 and parental CHO cells show identical DNA neutral filter elution dose-response: implications for a relationship between cell radiosensitivity and induction of DNA double-strand breaks.

The purpose of this work was to investigate a possible correlation between DNA elution dose-response and cell radiosensitivity. For this purpose neutral (pH 9.6) DNA filter elution dose-response curves were measured with radiosensitive xrs-5 and the parental Chinese hamster ovary (CHO) cells in the logarithmic and plateau phase of growth. No difference was observed between the two cell types in the DNA elution dose-response curves either in logarithmic or plateau phase, despite the dramatic differences in cell radiosensitivity. This observation indicates that the shape of the DNA elution dose-response curve and the shape of the cell survival curve are not causally related. It is proposed that the shoulder observed in the DNA elution dose-response curve reflects either partial release of DNA from chromatin, or cell cycle-specific alterations in the physicochemical properties of the DNA.

Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD.

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.

Differences in inhibition by beta-arabinofuranosyladenine (araA) of radiation induced DNA damage repair in exponentially growing and plateau-phase CHO-cells.

The effect of beta-arabinofuranosyladenine (araA) on the repair of radiation induced DNA damage, as measured by the DNA unwinding technique, was studied in exponentially growing and plateau-phase CHO-cells after exposure to x-rays. Induction of DNA damage by radiation was found to be similar in exponentially growing and plateau-phase cells. In the absence of araA, repair of radiation induced DNA damage proceeded with similar kinetics in exponentially growing and plateau-phase cells. AraA at concentrations between 0-1500 microM inhibited DNA repair both in exponentially growing and in plateau-phase cells. However, the degree of inhibition was significantly higher (by a factor of 3) in plateau-phase cells. A similar degree of repair inhibition by araA was observed in plateau phase cells treated in their conditioned medium, as well as in plateau phase cells that were transfered in fresh growth medium just before treatment initiation. These results indicate the importance of biochemical parameters associated with alterations in the growth state of the cells for the inhibitory effect of araA and may help in the elucidation of the molecular mechanism(s) underlying repair inhibition by inhibitors of DNA replication.

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells.

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.