Diagnosis and effects of urine contamination in cooled-extended stallion semen.

Urospermia is known to affect semen quality in many mammals, including stallions. Determinations of semen pH and creatinine and urea concentrations have been used to diagnose urine contamination in raw stallion semen. Unfortunately, practitioners suspecting urine contamination in cooled-shipped samples have no proven means to confirm the presence of urine. Therefore, the objectives of this study were (1) to assess the effects of urine contamination on sperm motility of extended fresh and cooled-stored stallion semen, (2) to evaluate the usefulness of semen color, odor, pH, and creatinine and urea concentrations for urospermia diagnosis, and (3) to evaluate the accuracy of a commercial blood urea nitrogen test strip in diagnosing urine contamination in extended-cooled stallion semen. Thirty-seven ejaculates were obtained from 11 stallions with no history of urospermia before division into 5 mL aliquots, and contamination with stallion urine. Each resulting sample was assessed for sperm motility, color, odor, pH, creatinine, and urea nitrogen concentration using both a semiquantitative test strip (Azostix), and a quantitative automated analyzer before and after cooling for 24 hour. Sperm motility parameters, pH, and creatinine and urea concentrations were analyzed using mixed models. Urine contamination decreased total and progressive motility in all samples before and after cooling (P < 0.05). Mean control total motility was 80% at 0 hour and 67% at 24 hours, whereas urine-contaminated samples ranged from 30% to 71% at 0 hour and 27% to 61% at 24 hours. Control mean urea (29 mg/dL) and creatinine (0.6 mg/dL) concentrations were significantly different (P < 0.05) from all urine-contaminated samples (158 mg/dL and 11.6 mg/dL, respectively) at 0 hour. Similarly, control mean urea (8 mg/dL) and creatinine (0.9 mg/dL) concentrations were significantly different than all urine-contaminated samples at 24 hours. Odor assessment presented moderate sensitivity (65%) and high specificity (100%), while color assessment presented low sensitivity (47%) and moderate specificity (79%) for urine in extended semen. Azostix strips were highly sensitive (95%) and specific (97%). Assessment of color, odor, and pH are not reliable methods to diagnose urine in experimentally contaminated cooled-stored stallion semen. Sperm motility parameters (in raw and cooled semen) are significantly reduced by the presence of urine in a concentration dependent. The results of the present study indicated that determination of urea and creatinine concentrations can be used to diagnose urospermia and that Azostix can be used as a point care method for diagnosing urine contamination in extended cooled stallion semen.

α-TEA cooperates with MEK or mTOR inhibitors to induce apoptosis via targeting IRS/PI3K pathways.

BACKGROUND: α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers. METHODS: Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques. RESULTS: α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them. CONCLUSIONS: Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

RRR-alpha-tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation.

RRR-alpha-Tocopheryl succinate (vitamin E succinate, VES) is a potent antitumor agent, inducing DNA synthesis arrest, differentiation, and apoptosis. Because little is known about VES-induced differentiation, studies reported here characterize VES effects on the differentiation status of human breast cancer cell lines and investigate possible molecular mechanisms involved. VES-induced differentiation of human MCF-7 and MDA-MB-435 breast cancer cells was characterized by morphological changes, induction of lipid droplets, induction of beta-casein mRNA expression, and down-regulation of Her2/neu protein. In contrast, VES treatment of normal human mammary epithelial cells, MCF-10A cells, and T-47D cells did not induce differentiation. Studies addressing mechanisms showed that neither antibody neutralization of the transforming growth factor-beta signaling pathway nor expression of a dominant-negative mutant of c-Jun N-terminal kinase blocked the ability of VES to induce differentiation; however, treatment of cells with PD 98059, a chemical inhibitor of mitogen-activated protein kinase kinase (MEK1/2), blocked the ability of VES to induce differentiation.

Activation of extracellular signal-regulated kinase and c-Jun-NH(2)-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells.

RRR-alpha-tocopherol succinate (vitamin E succinate, VES) is a potent, selective apoptotic agent for cancer cells but not normal cells. VES has been shown to inhibit the growth of a wide variety of tumor cells in cell culture and animal models. Studies addressing mechanisms of action of VES-induced apoptosis have identified transforming growth factor-beta, Fas/CD95-APO-1, and mitogen-activated protein kinase (MAPK) signaling pathway involvement. Here we show that MAPKs, the extracellular signal-regulated kinases (ERK), and the stress-activated protein kinases, c-Jun NH2-terminal kinases (JNK), but not p38, are critical mediators in VES-induced apoptosis of human breast cancer MDA-MB-435 cells. VES activates ERK1/2 and JNK both in level and duration of kinase activity. Expression of dominant negative mutants of ERK1, MAPK/ERK activator-1, or JNK1 but not p38 blocked phosphorylation of the substrate glutathione S-transferase-c-Jun and inhibited VES-induced apoptosis. Increased phosphorylation and transactivation activity of nuclear transcription factors c-Jun, ATF-2, and Elk-1 are observed after VES treatments; however, only c-Jun and ATF-2 appear to be involved in VES-induced apoptosis based on antisense blockage experiments. Collectively, these results imply a critical role for ERK1 and JNK1 but not p38 in VES-induced apoptosis of human MDA-MB-435 breast cancer cells.

Muscular dystrophy in female dogs.

The most common form of muscular dystrophy in dogs and humans is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome, and, therefore, disease-causing mutations in dystrophin occur most often in males. Therefore, females with dystrophin deficiency or other forms of muscular dystrophy may be undiagnosed or misdiagnosed. Immunohistochemistry was used to analyze dystrophin and a number of other muscle proteins associated with muscular dystrophy in humans, including sarcoglycans and laminin alpha2, in muscle biopsy specimens from 5 female dogs with pathologic changes consistent with muscular dystrophy. The female dogs were presented with a variety of clinical signs including generalized weakness, muscle wasting, tremors, exercise intolerance, gait abnormalities, and limb deformity. Serum creatine kinase activity was variably high. One dog had no detectable dystrophin in the muscle; another was mosaic, with some fibers normal and others partly dystrophin-deficient. A 3rd dog had normal dystrophin but no detectable laminin alpha2. Two dogs could not be classified. This study demonstrates the occurrence of dystrophin- and laminin alpha2-associated muscular dystrophy and the difficulty in clinical diagnosis of these disorders in female dogs.

From "reducing" to "coping with" uncertainty: reconceptualizing the central challenge in breast self-exams.

An ideology of uncertainty reduction pervades scholarly and popular discourse on breast self-exams (BSE). Women are encouraged to understand BSE as an activity that reduces uncertainty about their health. Moreover, uncertainties about the procedure itself are conceived as barriers to BSE. In turn, reducing these uncertainties is seen as the key to promoting BSE. We argue that the ideology of uncertainty reduction is both descriptively and prescriptively inadequate and potentially a threat to women's health. We further contend the ideology should be replaced by a framework that illuminates processes of coping with uncertainty. Several major characteristics of such a framework, as well as implications for medical practice, are discussed and illustrated within the context of BSE.

Breast self-examination pamphlets: a content analysis grounded in fear appeal research.

In this study, we used the topic of breast self-examination (BSE) to illustrate how content analysis of promotional texts (already in existence, in the process of being created, or both) can provide supplementary data to that derived from audience analysis. Specifically, we used content analysis to isolate messages in BSE pamphlets that are consistent with the variables of severity, susceptibility, response efficacy, and self-efficacy, identified by existing fear appeal research and supported by other persuasion research as critical to the construction of effective health promotion messages. We then used statistical analyses to describe the relation among these 4 message variables. Our findings suggested that BSE pamphlets contain an unbalanced proportion of threat to efficacy arguments. Additionally, the efficacy messages were substantively weak. We contrasted these messages against the relatively strong mammography arguments contained in these pamphlets. We then provided recommendations for formulating stronger persuasive arguments in BSE promotional materials.

Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis.

The apoptosis-triggering properties of vitamin E succinate (VES, RRR-alpha-tocopheryl succinate) for human LNCaP and PC-3 prostate carcinoma cells and normal PrEC human prostate epithelial cells were investigated. LNCaP and PC-3 cells were sensitive to VES-induced apoptosis, with 100% and 60% of cells undergoing apoptosis after three days of treatment with 10 micrograms of VES/ml, respectively. PrEC cells were resistant to VES-induced apoptosis. Treatment of prostate cells with agonistic anti-Fas antibody triggered apoptosis in approximately 50% of PC-3 cells within 48 hours, whereas LNCaP and PrEC cells were resistant. Prostate cells simultaneously treated with VES and agonistic anti-Fas antibodies revealed 1) no effect on PrEC cells, 2) an additive effect on Fas-sensitive PC-3 cells, and 3) a synergistic effect on LNCaP cells. VES treatment of LNCaP cells caused depletion of cytosolic 43-kDa Fas, enhanced membrane levels of 43-kDa Fas, and induced Fas sensitivity. PC-3 cells expressed high levels of membrane 43-kDa Fas that were enhanced by VES treatments. Fas ligand expression by LNCaP cells was enhanced by VES treatments. In summary, VES triggers apoptosis in human prostate carcinoma cells but not normal prostate cells in vitro, and VES modulates Fas signaling.

N-(4-hydroxyphenyl)retinamide activation of transforming growth factor-beta and induction of apoptosis in human breast cancer cells.

N-(4 hydroxyphenyl)retinamide (4-HPR), a synthetic derivative of all-trans-retinoic acid, induces DNA synthesis arrest and apoptosis in human breast cancer cells in a dose- and time-dependent manner. MDA-MB-435 cells treated with 3 microM 4-HPR exhibited 58% and 75% DNA synthesis arrest after 1 and 2 days of treatment and 31%, 39%, 48%, and 56% apoptosis after 3, 4, 5, and 6 days of treatment, respectively. Conditioned media from 4-HPR-treated MDA-MB-435 cells contained 63 and 57 pg of active transforming growth factor-beta (TGF-beta) per 10(6) cells after 1 and 2 days of treatment, whereas conditioned media from control cells contained only 9 pg/10(6) cells. TGF-beta involvement in 4-HPR-induced apoptosis, but not DNA synthesis arrest, in MDA-MB-435 cells was demonstrated by 1) blockage of 4-HPR-induced apoptosis by 66-75% after treatment of cells with neutralizing antibodies to TGF-beta s, 2) blockage of 4-HPR-induced apoptosis by 64-67% after transient transfection of cells with antisense oligomers to TGF-beta 1 or TGF-beta type II receptor, 3) blockage of 4-HPR-induced apoptosis by approximately 50% after inhibition of latent TGF-beta activation, and 4) demonstration that human breast cancer cells (T47D) defective in TGF-beta signaling were refractive to 4-HPR-induced apoptosis. These data indicate that 4-HPR is a potent activator of TGF-beta and that TGF-beta participates in 4-HPR-induced apoptosis of human breast cancer cells.

Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols.

The apoptosis-inducing properties of RRR-alpha-, beta-, gamma-, and delta-tocopherols, alpha-, gamma-, and delta-tocotrienols, RRR-alpha-tocopheryl acetate (vitamin E acetate), and RRR-alpha-tocopheryl succinate (vitamin E succinate) were investigated in estrogen-responsive MCF7 and estrogen-nonresponsive MDA-MB-435 human breast cancer cell lines in culture. Apoptosis was characterized by two criteria: 1) morphology of 4,6-diamidino-2-phenylindole-stained cells and oligonucleosomal DNA laddering. Vitamin E succinate, a known inducer of apoptosis in several cell lines, including human breast cancer cells, served as a positive control. The estrogen-responsive MCF7 cells were more susceptible than the estrogen-nonresponsive MDA-MB-435 cells, with concentrations for half-maximal response for tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol of 14, 15, 7, and 97 micrograms/ml, respectively. The tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol induced MDA-MB-435 cells to undergo apoptosis, with concentrations for half-maximal response of 176, 28, 13, and 145 micrograms/ml, respectively. With the exception of RRR-delta-tocopherol, the tocopherols (alpha, beta, and gamma) and the acetate derivative of RRR-alpha-tocopherol (RRR-alpha-tocopheryl acetate) were ineffective in induction of apoptosis in both cell lines when tested within the range of their solubility, i.e., 10-200 micrograms/ml. In summary, these studies demonstrate that naturally occurring tocotrienols and RRR-delta-tocopherol are effective apoptotic inducers for human breast cancer cells.

Chronic modulation of the TCR repertoire in the lymphoid periphery.

Using TCR V beta 5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+V beta 5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+V beta 5+ T cells transit through a CD8lowV beta 5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4-CD8-V beta 5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V alpha repertoire of each of these cellular compartments in both V beta 5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V alpha families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.

Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis.

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.

Hemodynamic effects of thyroidectomy in sedentary horses.

OBJECTIVE: To investigate hemodynamic effects of thyroidectomy in horses at rest. ANIMALS: 6 healthy aged Quarter Horse mares. PROCEDURE: Horses were monitored for 5 months before and 4 weeks after thyroidectomy and for an additional 4 weeks after administration of thyroid hormone supplement (2.5 microg of thyroxine/kg of body weight, PO, q 12 h, and 0.6 microg of triiodothyronine/kg, PO, q 12 h). Responses to thyroid-stimulating hormone (TSH) were measured before and 4 weeks after thyroidectomy. Other variables monitored daily were resting rectal temperature (T), heart rate (HR), respiratory rate (RR), and body weight (BW). Monthly cardiac output (Q), blood volume (BV), plasma volume (PV), standard electrocardiographic measures, systolic and right ventricular blood pressure, and HR responses were determined after IV administration of isoproterenol and phenylephrine. Variables were analyzed by use of repeated-measures ANOVA. RESULTS: Complete thyroidectomy was confirmed by minimal response to TSH 4 weeks after surgery. Resting HR, RR, T, Q, and beta-adrenergic responsiveness to isoproterenol decreased significantly after thyroidectomy. Resting T, Q, and beta-adrenergic responsiveness increased after administration of supplement and was not significantly different from euthyroid values. Blood volume and PV increased significantly after thyroidectomy but did not return to euthyroid values despite administration of supplement. Response to phenylephrine was minimally different between treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Thyroidectomy in horses caused decreased resting HR, RR, T, Q, and isoproterenol responsiveness and increased BV, PV, PQ interval, and QT interval corrected for HR. Some of these surgically induced changes appeared to be partially reversed by administration of thyroid hormone supplement.

Reading and reforming breast self-examination discourse: claiming missed opportunities for empowerment.

For close to 45 years breast self-examination (BSE) has had nearly uncontested support as depicted in mass media accounts. Just recently, however, a number of breast cancer activists have spoken out against the practice. They claim that BSE is oppressive and fosters victim blaming, which simply is not in the best interests of women. Judging by the media reports I reviewed, these activists may have a valid point. The discourse of the popular media articles that constitutes the issue of BSE for the average woman blames women for not doing their part to reduce high breast cancer mortality statistics, establishes the locus of all reasons for refraining from the activity with the woman, and chastises these women for failing to engage in the activity. Moreover, in addition to being an agency-robbing discourse, these media reports provide minimal (if any) substantive rationale that there is any benefit to BSE. It appears, then, that women are subjected to an agency-robbing discourse and, as it is currently enacted, practice for what appears to be little reason. However, this does not mean that BSE is an inefficacious means of early detection or that women cannot be empowered by participating in the discursive process and the physical act itself. Rather, in the discussion of this essay I provide a number of suggestions for reforming BSE rhetoric such that it facilitates a more therapeutic and individually empowering self-help activity.

Absence of longitudinal changes in rheumatologic parameters after silicone breast implantation: a prospective 13-year study.

There have been numerous questions regarding the association of polysiloxane with connective tissue disease and alteration of host immune response. C-reactive protein, rheumatoid factor, and anti-streptolysin-O titers were measured in 218 patients. These studies are routinely used in the diagnosis of autoimmune disease and mixed connective tissue disease. This prospective study has been in progress since 1985. The first patients were seen in July of 1985, and those individuals willing to participate were followed from 1985 to 1998. The implants included saline-filled elastomer shells and polysiloxane gel-filled elastomer shells. These groups were examined separately and in combination for changes between preoperative and postoperative states. In each instance, there was no statistical increase or decrease. Each patient underwent a physical examination and completed a questionnaire focusing on signs and symptoms of autoimmune and connective tissue diseases. The laboratory data and subjective clinical results demonstrated no significant differences between a nonimplanted group versus the saline group alone, the gel group alone, or the combined groups. The data failed to suggest any causal relationship between implants and autoimmune or connective tissue diseases over the study period of 13 years (since 1985).

Feline epilepsy.

The diagnosis and management of seizures in the cat require an understanding of the more common diseases that predispose to feline epilepsy. Feline seizures may occur secondary to intracranial or extracranial disease. Intracranial causes include inflammatory, neoplastic, vascular, and traumatic disorders. Extracranial causes include various metabolic and toxic insults. Previous brain insults that are no longer active may leave "seizure foci." Idiopathic epilepsy is uncommon in the cat relative to the dog but should be considered if no cause can be found. Regardless of the etiology, ictal events in cats can manifest themselves in multiple forms and levels of severity. Therapy should be directed at controlling seizure frequency and treating the underlying cause. An aggressive diagnostic and therapeutic approach to feline epilepsy may improve prognosis and lead to a favorable outcome.

RRR-alpha-tocopheryl succinate induction of prolonged activation of c-jun amino-terminal kinase and c-jun during induction of apoptosis in human MDA-MB-435 breast cancer cells.

We have demonstrated that RRR-alpha-tocopheryl succinate (10 microg/mL vitamin E succinate (VES) treatment of estrogen receptor-negative MDA-MB-435 human breast cancer cells induces 9, 19, 51, and 72% apoptotic cells on days 1-4, respectively, after treatment, which involves transforming growth factor-beta signaling. Here, we show that VES-triggered apoptosis of MDA-MB-435 cells induced prolonged elevated expression of c-jun mRNA and protein (neither of which was caused by major increases in stability) and also induced enhanced activator protein-1 (AP-1) binding to the consensus DNA oligomer. Furthermore, VES treatments resulted in increased AP-1 transactivation activity, as measured with an AP-1 promoter/luciferase reporter construct and by the measurement of increased mRNA expression of the AP-1-dependent endogenous gene collagenase. Evidence of VES-induced involvement of the c-jun amino-terminal kinase in these AP-1-dependent events was suggested by data showing prolonged activity of this kinase, as measured by a kinase assay using glutathione S-transferase-c-jun as the substrate. The c-jun-dependent transcriptional activity was verified by cotransfection of a chimeric transcription factor having a galactose 4 DNA-binding domain coupled with the transactivation domain of c-jun plus the reporter plasmid 5X GAL4-luciferase. MDA-MB-435 cells infected with an adenovirus expression vector containing the TAM-67 sequence for dominant/negative-acting mutant c-jun or transiently transfected with c-jun antisense exhibited a 50-77% reduction in VES-mediated apoptosis as compared with control adenovirus-infected or control sense oligomer-transfected cells.

c-Jun involvement in vitamin E succinate induced apoptosis of reticuloendotheliosis virus transformed avian lymphoid cells.

Previous studies have shown that treatment of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells with 10 microg/ml (18.8 microM) of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for 3 days induced approximately 50% of the cells to undergo apoptosis. Elevated and prolonged expression of c-jun mRNA and protein was temporally correlated with VES-induced cell death. Data presented in this paper show that the elevated and prolonged expression of c-jun message and protein are not accounted for by enhanced stability, and show the involvement of c-Jun in VES-induced apoptosis in this lymphoblastoid cell type. C4-1 cells infected with a virus carrying a dominant, negatively acting mutant form of c-Jun, supjun-1, exhibited: (i) 71% reduction in VES-induced apoptosis, (ii) a 2.0-2.5-fold decrease in wildtype, endogenous c-Jun expression, and (iii) a 2.4-2.6-fold reduction in AP-1 binding activity. Additionally, cells co-treated with VES plus RRR-alpha-tocopherol, exhibited a 70% reduction in apoptosis, a marked reduction in c-Jun expression and a 1.6-fold reduction in AP-1 binding activity. These studies suggest that c-Jun plays a crucial role in VES-induced apoptosis in C4-1 cells, and add to our understanding of mechanisms of action involved in VES-mediated tumor cell growth inhibition.

Involvement of activator protein-1 (AP-1) in induction of apoptosis by vitamin E succinate in human breast cancer cells.

The purpose of this study was to document induction of apoptosis by vitamin E succinate (VES; RRR-alpha-tocopheryl succinate) in human breast cancer cells in culture and to characterize potential c-jun involvement. VES at 18.8 microM (10 micrograms/mL) induced DNA synthesis arrest, reduced total cell numbers, and induced apoptosis in estrogen receptor-positive and estrogen-responsive MCF-7 human breast cancer cells. VES at 10 micrograms/mL induced apoptosis in greater than 60% of cells within 3 d of treatment. Apoptosis was documented by detection of fragmented or condensed nuclei in 4',6-diamindino-2-phenylindole-stained cells, detection of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeled DNA, and DNA laddering. Analyses of mRNA and protein levels of candidate molecules involved in apoptosis showed that MCF-7 cells treated with VES exhibited elevated and persistent expression of c-jun. MCF-7 cells stably transfected with a dominant-negative interfering mutant c-jun, TAM-67, and expressing high levels of mutant jun exhibited approximately 50% blockage of VES-mediated apoptosis. In addition to increased c-jun expression after VES treatment, VES-treated MCF-7 cells exhibited elevated activator protein-1 (AP-1) binding activity. Comparisons of AP-1 binding factors by super-shift analyses with jun-specific antibodies in cells sensitive to VES-induced apoptosis (empty-vector control 7-1 cells) and cells resistant to VES-induced apoptosis (TAM-67-containing TAM-9 cells) showed that the sensitive cells expressed c-jun and jun D and the resistant cells TAM-67 AP-1 binding proteins after VES treatment. These studies suggested that c-jun may be involved in the apoptotic process initiated by VES treatment of human MCF-7 breast cancer cells.