Evaluation of the expression and intracellular localization of a 44-kDa calmodulin binding protein during exponential growth and quiescence (G0).

We have previously demonstrated that changes in calmodulin (CaM) levels are associated with G1/S transition of the cell cycle and entry into and release from quiescence (G0). CaM mediates its regulation through the specific interaction with different intracellular proteins called calmodulin binding proteins (CaMBPs). This study was designed to evaluate the expression of the CaMBPs during the cell cycle. Mouse C127 cells were synchronized in quiescence (G0) by serum deprivation. Analysis of the CaMBPs by the 125I-labeled CaM ([125I]CaM) overlay procedure on one- and two-dimensional gels revealed many proteins that bind to CaM at any given time during the cell cycle. However, specific expression of a 44-kiloDalton CaMBP (44CaMBP) was observed. As cells entered quiescence (G0) phase, there was a decrease in the CaM binding to the 44CaMBP. During release into the cell cycle from G0 phase, the binding to CaM was maintained at the low level, but reappeared as the cells entered S phase. CaM binding to the 44CaMBP was intense during S phase and decreased as the cells progressed into G2/M. Antibody directed against the 44CaMBP was produced in rabbit. Quantitation of the 44CaMBP by Western blot analysis revealed a similar pattern to that observed by the [125I]CaM overlay procedure during the course of G0 entry and release. The anti-44CaMBP antibody was used to evaluate the intracellular localization of the 44CaMBP by indirect immunofluorescence. A distinctive punctate nuclear staining, Mwas observed. This punctate nuclear staining, observed in all cells during exponential growth, disappeared as the cells entered G0. The nuclear staining remained absent in cells released from G0 until the cells approached and entered the S phase, at which time the punctate nuclear staining reappeared. This staining pattern was then maintained through G2/M progression. Following M phase and entry into G1 phase, the punctate nuclear staining was observed in all G1 cells. Similar analysis for cells synchronized at the G1/S boundary by the double thymidine block procedure revealed that the punctate nuclear staining was present in all cells throughout the entire course of the cell cycle. The immunofluorescence staining pattern for the 44CaMBP was sensitive to the anti-CaM drug W13 at a dose that is known to reversibly block cells at G1/S. No effect was observed by the inactive analog W12. The punctate nuclear staining of the 44CaMBP would appear to be present during all phases of the cell cycle when cells are committed to be in the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo.

Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.

Decrease in calmodulin concentrations during heparin-induced capacitation in bovine spermatozoa.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

The phospholipase A2 of human spermatozoa; purification and partial sequence.

In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution.

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.

Increased sensitivity to Vinca alkaloids in cells overexpressing calmodulin by gene transfection.

Mouse C127 cells, transfected with the chicken calmodulin (CaM) gene and overexpressing CaM protein, were used to evaluate the effect of elevated levels of CaM on the sensitivity of these cells to various anticancer drugs. Clones C2 and C3 overexpress CaM mRNA by 40- and 80-fold, respectively, and CaM protein 3- and 8-fold, respectively. These cell lines were tested for their sensitivity to vincristine, vinblastine, bleomycin, and Adriamycin by comparing the 50% inhibitory concentration in a 72-h growth inhibition assay. The 50% inhibitory concentration values for vincristine with C2 and C3 cells were 6.27 +/- 0.56 nM and 6.60 +/- 0.96 nM, respectively. These values were significantly lower than 13.9 +/- 0.79 nM for the parental C127 cells and 14.0 +/- 1.55 nM for clone 6.8 (the control cell line for transfection without the chicken CaM gene) at P less than or equal to 0.005. The proliferation of C2 and C3 cells was inhibited at lower concentrations of vinblastine as well. The 50% inhibitory concentration values for the C2 and C3 cell lines were approximately one-half those required for C127 or clone 6.8 cells. However, no significant difference in the sensitivity to the DNA-binding drugs, bleomycin and Adriamycin, was observed between the different cell lines. The uptake of [3H]vinblastine was evaluated and found to be increased 1.6- and 2.8-fold in C2 and C3 cells, respectively, as compared with that value obtained for C127 cells. Moreover, the efflux of [3H]vinblastine from vinblastine-loaded cells was also observed to be decreased in the C2 and C3 cell lines. These data suggest that the increase in CaM expression in the C2 and C3 cell lines might be related to the higher sensitivity of these cells to Vinca alkaloids. This increased sensitivity appears to be due to the increase in intracellular concentration of the Vinca alkaloids as a result of an increase in drug uptake and a decrease in efflux. Moreover, the increased sensitivity of clones C2 and C3 to Vinca alkaloids appears to be specific to this class of drugs in that no collateral effects were observed for the DNA-damaging drugs, Adriamycin and bleomycin.

Identification of a 51-kilodalton calmodulin binding protein that changes during estrogen-stimulated cell growth.

Calmodulin-binding proteins (CaMBPs) were analyzed during estrogen-stimulated growth in the human breast cancer cell line ZR-75-1. A variety of Ca2(+)-dependent and -independent CaMBPs were observed to be present in these cells. Calmodulin (CaM) binding to a 51-kilodalton protein was shown to be Ca2(+)-dependent. Moreover, binding to this protein was reduced in the estrogen-treated cells. This effect occurred early during estrogen-stimulated cell growth and was maintained during exponential growth in the presence of estrogen. 125I-labeled CaM overlay procedure of two-dimensional polyacrylamide gels reveals that this 51-kilodalton protein is composed of at least two distinct isoforms with different isoelectric points. Subcellular localization demonstrates that this protein resides exclusively in the microsomal fraction.

Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation.

The 125I-calmodulin gel overlay procedure was used to evaluate the effect of a heparin treatment on the calmodulin-binding proteins of bull spermatozoa. At concentrations that increase the in vitro fertilization rate of in vitro-matured oocytes, heparin induced a decrease in the binding to calmodulin (CaM) in 3 sperm proteins of 28, 30, and 49 kDa. The binding of these proteins to CaM was higher when Ca2+ was absent from the overlay procedure, and this binding was negatively correlated to the fertilization rate. These results suggest that sperm capacitation is associated with a decrease in the binding of CaM to the 28, 30, and 49 kDa sperm CaM-binding proteins. Implications of such a decrease are discussed.

In vitro interactions of calmodulin with the ovine proacrosin-acrosin system.

The authors studied the interaction of calmodulin (CaM) with proacrosin and acrosin from ram spermatozoa. CaM binding evaluated by the [125I]-CaM overlay procedure was shown to occur preferentially with both proacrosin and acrosin in the presence of EGTA; in the presence of Ca2+, the interaction was less intense. Further studies with native proenzyme preparations showed that proacrosin activation at pH 7.1 or 8.0 was significantly accelerated in the presence of CaM and EGTA (t1/2 = 23 min vs. 55 min for EGTA alone at pH 7.1), but not in the presence of Ca2+ (t1/2 = 73 min). The enzymatic activity of acrosin towards benzoyl arginine paranitroanilide, however, was not significantly affected by CaM whether Ca2+ was absent or present. Finally, the authors demonstrated that acrosin hydrolyzed CaM rapidly and extensively in the presence of EGTA. These results indicate that CaM interacts in vitro with proacrosin and acrosin, and that acrosin can attenuate CaM activity through proteolysis. Whether these interactions also occur in vivo and are involved in some aspects of spermatozoa function remains to be determined.

Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa.

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.

Developmental appearance of the Ca2+-binding proteins parvalbumin, calbindin D-28K, S-100 proteins and calmodulin during testicular development in the rat.

Calcium and intracellular Ca2+-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Potentiation of bleomycin lethality by anticalmodulin drugs: a role for calmodulin in DNA repair.

Treatment of exponentially growing Chinese hamster ovary cells with bleomycin causes a dose-dependent decrease in cell survival due to DNA damage. This lethal effect can be potentiated by the addition of a nonlethal dose of the anticalmodulin drug N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide ( W13 ) but not its inactive analog N-(4-aminobutyl)-2-naphthalenesulfonamide ( W12 ). By preventing the repair of damaged DNA, W13 also inhibits recovery from potentially lethal damage induced by bleomycin. These data suggest a role for calmodulin in the DNA repair pathway.

Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells.

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle.

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.

Calmodulin-binding proteins in a cloned rat insulinoma cell line.

Calmodulin concentrations were measured in isolated hamster islets or in a cloned rat insulin-secreting cell line RIN-m-5F treated with high glucose. There was no change in the cellular calmodulin content of either islets or RIN-m-5F cells despite increases of insulin concentrations in the media. Treatment of cells with the anti-calmodulin drug W13 inhibited insulin-stimulated glucose release, whereas a small effect on insulin accumulation in the media was observed with W12, the dechlorinated, less active analogue of W13. At the lowest dose tested (30 microM) the effect of W13 on insulin accumulation in the media was completely reversible. To further investigate the possible role of calmodulin in insulin secretion, calmodulin-binding proteins in subcellular fractions of the RIN-m-5F cells were identified using a gel overlay technique. Ca2+-dependent binding of 125I-calmodulin was observed to cytosolic proteins with apparent Mr = 125, 110, 56, 52, and 34 k (kilodalton). This binding was completely displaceable with unlabeled calmodulin, whereas only partial displacement was observed when the homologous Ca2+ binding protein of skeletal muscle, troponin C, was used. Four proteins with Mr similar to the histones bind calmodulin in a Ca2+-independent manner. 125I-calmodulin:calmodulin binding protein interactions were inhibited in a dose-dependent manner by the anti-calmodulin drug, W13. Little effect was observed with the analogue W12.(ABSTRACT TRUNCATED AT 250 WORDS)

Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression.

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle unit cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.