Terminal Deoxynucleotidyl Transferase-mediated dUTP-biotin End Research Articles

Artemisiae Iwayomogii Herba Inhibits Growth, Motility, and the PI3K/AKT/mTOR Signaling Pathway in Hepatocellular Carcinoma Cells.

Artemisia gmelinii (Artemisia iwayomogi) has been used in traditional medicine to cure various infectious diseases such as cholecystitis, hepatitis, and jaundice. In this study, the Artemisiae Iwayomogii Herba ethanol extract was investigated for the ability to inhibit growth of hepatocellular carcinoma and its underlying mechanism involved. The antiproliferative effect of Artemisiae Iwayomogii Herba ethanol extract was evaluated using cell viability and proliferation assays. The effect of Artemisiae Iwayomogii Herba ethanol extract on apoptosis was measured using western blotting, terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining, JC-1 staining, cytochrome c release, immunohistochemistry, and immunofluorescence in ex vivo mouse xenografts. Artemisiae Iwayomogii Herba ethanol extract inhibited hepatocellular carcinoma cell growth and proliferation in a dose-dependent manner. The apoptotic effect of Artemisiae Iwayomogii Herba ethanol extract was observed via increased levels of cleaved caspase-3 and cleaved PARP, as well as elevated numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling-positive apoptotic cells. Artemisiae Iwayomogii Herba ethanol extract also decreased XIAP and Mcl-1 expression via loss of mitochondrial membrane potential. Additionally, Artemisiae Iwayomogii Herba ethanol extract inhibited hepatocellular carcinoma cell invasion and migration. In the ex vivo model, Artemisiae Iwayomogii Herba ethanol extract significantly inhibited tumor cell proliferation and increased the number of apoptotic cells with more activated cleaved caspase-3. A mechanistic study revealed that Artemisiae Iwayomogii Herba ethanol extract effectively suppressed the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma cells. Our findings demonstrate that Artemisiae Iwayomogii Herba ethanol extract can efficiently induce apoptosis and inhibit the growth, migration, and invasion of human hepatocellular carcinoma cells, and simultaneously block PI3K/AKT/mTOR pathway. We therefore suggest Artemisiae Iwayomogii Herba ethanol extract as a novel natural agent for prevention and therapy of hepatocellular carcinoma.

Apoptotic Effects of Xanthium strumarium via PI3K/AKT/mTOR Pathway in Hepatocellular Carcinoma

Xanthium strumarium (XS) has been traditionally used as a medicinal herb for treating inflammatory diseases, such as appendicitis, chronic bronchitis, rheumatism, and rhinitis. In this study, we yielded ethanol extracts from XS and investigated whether they could inhibit the progression of hepatocellular carcinoma (HCC) and its underlying mechanism. The XS-5 and XS-6 extracts dose-dependently inhibited the growth and proliferation in HCC cell lines. The apoptotic effects of them were observed via increased levels of cleaved caspase-3 and cleaved PARP, as well as elevated numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling- (TUNEL-) positive apoptotic cells. They also decreased XIAP and Mcl-1 expression via loss of mitochondrial membrane potential. Additionally, they inhibited the invasion and migration of HCC cells. In an ex vivo model, the extracts significantly inhibited tumor cell growth and induced apoptosis by increasing the expression of the cleaved caspase-3. A mechanistic study revealed that they effectively suppressed PI3K/AKT/mTOR signaling pathways in HCC cells. Taken together, our findings demonstrate that they could efficiently not only induce apoptosis but also inhibit cell growth, migration, and invasion of human HCC cells by blocking the PI3K/AKT/mTOR pathway. We suggest XS-5 and XS-6 as novel natural anti-HCC agents.

Pitavastatin reduces oxidative stress and attenuates intermittent hypoxia-induced left ventricular remodeling in lean mice

We have reported previously that intermittent hypoxia related to sleep apnea induces cardiovascular remodeling secondary to the oxidative stress. The aim of this study was to examine the effect of pitavastatin as an antioxidant to prevent intermittent hypoxia-induced left ventricular (LV) remodeling in mice without hypercholesterolemia. Eight-week-old male C57BL/6J mice (n=35) were exposed to intermittent hypoxia (30 s exposure to 5% oxygen, followed by 30 s exposure to 21% oxygen) for 8 h per day during the daytime or maintained under normoxic conditions; in addition, they were either treated with pitavastatin (3 mg kg(-1) per day) or vehicle for 10 days. After cardiac catheterization and blood sampling, the LV myocardium was examined. The systemic blood pressure and plasma level of total cholesterol were similar among the four groups. Intermittent hypoxia significantly increased the expression levels of 4-hydroxy-2-nonenal (4-HNE) proteins, TNF-alpha and TGF-beta mRNA, and also the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL)-positive myocardial cells in the LV myocardium. In addition, enhanced hypertrophy of the cardiomyocytes, perivascular fibrosis and histological degeneration were observed in the mice exposed to hypoxic stress. Treatment with pitavastatin significantly suppressed the expression levels of the 4-HNE proteins, cytokines, superoxide production and TUNEL-positive myocardial cells in the LV myocardium, consequently attenuating the hypoxia-induced histological changes. Pitavastatin preserved, at least partially, the morphological structure of the LV myocardium in lean mice exposed to intermittent hypoxia, through its antioxidant effect.

Minocycline Inhibition of Photoreceptor Degeneration

To determine whether systemic minocycline can protect photoreceptors in experimental retinal detachment (RD). Retinal detachment was induced in mice by subretinal injection of sodium hyaluronate, 1.4%. In 1 experiment, mice received daily injections of minocycline (group 1) or saline (group 2). In a second experiment, mice were treated with minocycline or saline beginning 24 hours prior, immediately after, or 24 hours after experimental RD. In both experiments, photoreceptor cell survival and apoptosis were assessed by immunohistochemistry with primary antibodies against photoreceptor cell markers, rod rhodopsin, and cone opsin, and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Photoreceptor cell apoptosis was detected at day 1 after experimental RD, with apoptotic cells peaking in number at day 3 and dropping by day 7. Treatment with minocycline significantly reduced the number of apoptotic photoreceptor cells associated with RD when given 24 hours before or even 24 hours after RD. Our data suggest that minocycline may be useful in the treatment of photoreceptor degeneration associated with RD, even when given up to 24 hours after RD. Use of minocycline in patients with macula-off RD may prevent photoreceptor apoptosis and glial cell proliferation, improving final visual outcomes.

GLYCYRRHIZIN REDUCES SECONDARY INFLAMMATORY PROCESS AFTER SPINAL CORD COMPRESSION INJURY IN MICE

Glycyrrhizin, a major active constituent of liquorice root (Glycyrrhiza glabra), has a free radical scavenging property, and its effects were evaluated on an animal model of spinal cord injury (SCI) induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling staining, Bax, and Bcl-2 expression). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS, and poly(adenosine diphosphate-ribose) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB. In contrast, the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) nitrotyrosine and poly(adenosine diphosphate [ADP] ribose) formation, (3) iNOS expression, (4) nuclear factor-kappaB activation, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling, Bax, and Bcl-2) was markedly reduced in spinal cord tissue obtained from mice treated with glycyrrhizin extract (10 mg/kg, i.p., 30 min before and 1 and 6 h after SCI). In a separate set of experiments, we have clearly demonstrated that glycyrrhizin extract treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with glycyrrhizin extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate‐associated osteonecrosis

Osteonecrosis of the jaw secondary to bisphosphonate infusion (zoledronic acid-ZA) is assumed to be a bone disease. This study investigated the effects of ZA on soft tissues using oral mucosal cells as an in vitro model of soft tissue cell death in the pathogenesis of bone necrosis. Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations of ZA (0.25-3 micromol/l), using 1 micromol/l as the expected baseline concentration. A dose-response effect on apoptosis and cell proliferation [Terminal deoxynucleotidyl transferase-mediated dUTP-Biotin End Labelling and Annexin V or Coulter counter and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), respectively] was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Gene expression analysis using RT(2) Profiler polymerase chain reaction Arrays demonstrated the differential expression of multiple genes involved in apoptosis including those that encode TNF, BCL-2, Caspase, IAP, TRAF and Death Domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression. This study demonstrated that low concentrations of ZA rapidly and directly affected the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings support the potential for soft tissue injury as an initiating/potentiating event for osteonecrosis.

The effects of NK4 on viral myocarditis mice

NK4 may be a promising agent to inhibit tumor invasion and metastasis. To observe the effects of NK4 on the cardiovascular system with pathological injury and to discuss the mechanism, we established an experimental model of viral myocarditis (VCM) by coxsackievirus B3 infection in Balb/c mice on Day 0 and administered NK4 twice daily to the VCM and control mice from Day 20 to Day 45. We then evaluated the cardiac function by means of ultrasonic inspection. Hepatocyte growth factor, TNF (tumor necrosis factor)-alpha, and angiotensin II levels in the myocardial tissue were measured with enzyme-linked immunosorbent assay. Myocardium histopathology was examined with hematoxylin and eosin stain. Collagen deposition of the myocardium was detected through Masson staining. Microvessel staining with the RECA antibody and apoptosis detection with terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling were performed in the myocardium. The changes in MMP3 (matrix metalloproteinase 3), MMP9, TIMP1 (tissue inhibitor of metalloproteinase 1), and TGF (transforming growth factor)-beta1 expression in the myocardium were measured by reverse-transcriptase polymerase chain reaction. We found that NK4 intervention increased TGF-beta and angiotensin II expression, suppressed MMPs, improved the activities of TIMPs, and then promoted collagen deposition in the myocardium. NK4 intervention also decreased the microvessels' density and increased the apoptotic cell count in the myocardia of VCM mice. However, we did not observe the obvious changes in the myocardia of control mice after NK4 intervention. These data suggest that NK4 made negative impacts on the restoration of cardiac function and the recovery from VCM in the experimental mice.

Impaired Stria Vascularis in the Inner Ear of Apolipoprotein E Gene Knockout Mice

Purpose: To study apoptotic cell death, the expression of endothelial nitric oxide synthase (eNOS) and the capillary density in the cochleae of apolipoprotein E gene knockout (ApoE KO) mice. Methods: Cochleae of ApoE KO mice were stained by HE, anti-caspase-3 and anti-eNOS antibodies and TUNEL (in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling). Capillary density was measured in the stria vascularis and in the modiolus. Results: Apoptotic cell death was found in the stria vascularis of ApoE KO mice, but not in the organ of Corti. The expression of eNOS and the capillary density were decreased in the stria vascularis and in the modiolus of ApoE KO mice. Conclusion: These results suggest that cells in the inner ear of ApoE KO mice take different pathways to undergo cell death, and apoptotic cell death is only involved in the stria vascularis. These pathological changes may play a role in the hearing loss that has been found in ApoE KO mice.

The ErbB Inhibitors Trastuzumab and Erlotinib Inhibit Growth of Vestibular Schwannoma Xenografts in Nude Mice

To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts. Vestibular schwannoma xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor trastuzumab or saline for 2 weeks. Animals also received bromodeoxyuridine injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging during the treatment period. Cell death was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA. Tumors can be distinguished with T2-weighted magnetic resonance imaging sequences. Trastuzumab significantly reduced the proliferation of VS cells compared with control (p < 0.01) as analyzed by bromodeoxyuridine uptake. Control tumors demonstrated slight growth during the 12-week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p < 0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA-positive VS cells (p < 0.01). In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice, raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis Type 2.

Multidrug Resistance-Associated Protein–Overexpressing Teniposide-Resistant Human Lymphomas Undergo Apoptosis by a Tubulin-Binding Agent

Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondrially-mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis-regulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3'-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3'-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.

Expression of DNA Damage Checkpoint Protein Hus1 in Epithelial Ovarian Tumors Correlates With Prognostic Markers

Hus1 and Rad9 are proteins involved in DNA damage checkpoint regulation, which is required for the maintenance of genomic stability. In addition to checkpoint activation, mammalian cells also use apoptosis to eliminate cells with severe DNA damage. Interestingly, Rad9 was shown to be directly involved in apoptosis as well. Despite the knowledge of molecular mechanisms on how Hus1 and Rad9 act in response to DNA damage, little is known about the role of these 2 proteins in cancer progression. In this study, we analyzed the expression of Rad9 and Hus1 in epithelial ovarian tumors and correlated them to clinopathological parameters and apoptotic biomarkers (p53, Bcl-2, and Bax). Histological sections from 114 primary ovarian epithelial tumors were stained with antibodies using the streptavidin-biotin method. In addition, mitotic and apoptotic indices (both hematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling assay) were also measured. We found that Rad9 expression correlated closely to significance only with the apoptotic and mitotic indices (P = 0.056 and 0.059, respectively). Hus1 levels correlated significantly with the clinicopathologic factors of bad prognosis, including FIGO (International Federation of Gynecology and Obstetrics) stage (P < 0.002) and with the p53 expression (P < 0.001), Bax expression (P < 0.008), mitotic index (P < 0.001), and apoptotic index (P < 0.003).

Effects of prenatal vitamins A, E, and C on the hypoplastic hearts of fetal rats with diaphragmatic hernia

Background/Aim Nitrofen induces heart hypoplasia together with congenital diaphragmatic hernia (CDH) in rats. Intracellular oxidative stress might be one of the mechanisms of action of the teratogen, and vitamin A has been shown to reverse in part these effects when administered simultaneously or shortly after it. This study aims at testing the hypothesis that vitamin A and other antioxidant vitamins, such as E and C, could improve myocardial development even when administered late in gestation, a likely useful period for prenatal medication. Material and Methods Time-mated Sprague-Dawley female rats were exposed to either vehicle (control) or 100 mg of nitrofen (experimental) on day 9.5 of gestation. In 3 additional groups, the animals were exposed to vitamin A (total 15 000 IU), vitamin E (total 150 IU), or vitamin C (total 150 IU) on days 16, 17, and 18. The fetuses were recovered on day 21, and randomly selected hearts of those with CDH were processed for histologic studies (hematoxylin-eosin and periodic acid-Schiff stainings), DNA and protein contents, and ki-67 (proliferation) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (apoptosis) studies. The differences among groups were assessed by analysis of variance with Bonferroni/Dunn post hoc tests and a threshold of significance of P < .05. Results Nitrofen induced heart hypoplasia in terms of decreased heart/body weight, cell mass (less DNA and protein), and proportion of proliferating cells with increased apoptosis. Vitamin C alleviated weight hypoplasia and the 3 vitamins were able to restore cell mass and to reestablish near-normal figures of proliferation and apoptosis. Conclusions Antioxidant vitamins A, E, and C given late in gestation alleviate heart hypoplasia that accompanies CDH in the rat model. This timing suggests that the beneficial effects are exerted on the maturational phase of development.

Elevated serum creatine phosphokinase in choline-deficient humans: mechanistic studies in C2C12 mouse myoblasts

Choline is a required nutrient, and humans deprived of choline develop liver damage. This study examined the effect of choline deficiency on muscle cells and the release of creatine phosphokinase (CPK) as a sequela of that deficiency. Four men were fed diets containing adequate and deficient amounts of choline, and serum was collected at intervals for measurement of CPK. C2C12 mouse myoblasts were cultured in a defined medium containing 0 or 70 micromol choline/L for up to 96 h, and CPK was measured in the media; choline and metabolites were measured in cells. Apoptosis was assessed by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling and activated caspase-3 immunohistochemistry. Cell fragility in response to hypo-osmotic stress was also assessed. Three of 4 humans fed a choline-deficient diet had significantly elevated serum CPK activity derived from skeletal muscle (up to 66-fold; P < 0.01) that resolved when choline was restored to their diets. Cells grown in choline-deficient medium for 72 h leaked 3.5-fold more CPK than did cells grown in medium with 70 micromol choline/L (control medium; P < 0.01). Apoptosis was induced in cells grown in choline-deficient medium. Phosphatidylcholine concentrations were diminished in choline-deficient cells (to 43% of concentrations in control cells at 72 h; P < 0.01), as were concentrations of intracellular choline, phosphocholine, and glycerophosphocholine. Cells grown in choline-deficient medium had greater membrane osmotic fragility than did cells grown in control medium. Choline deficiency results in diminished concentrations of membrane phosphatidylcholine in myocytes, which makes them more fragile and results in increased leakage of CPK from cells. Serum CPK may be a useful clinical marker for choline deficiency in humans.

Loss of bag-1 immunoreactivity in rat brain after transient middle cerebral artery occlusion

Although bag-1 is a strong apoptosis repressor protein, its functions in normal or injured brains are not fully understood. In the present study, we investigated expression of bag-1 protein in rat brain after transient middle cerebral artery (MCA) occlusion, and compared the results with that of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL). Immunohistochemical analysis revealed that neuronal, choroid plexus, and ependymal cells were positively stained in the sham control brain. After 90 min of transient MCA occlusion, immunoreactivity for bag-1 progressively decreased from 3 to 48 h in the nuclei of neurons. Western blot analysis revealed that immunoreactive bag-1 was markedly decreased in the nuclear fraction. In contrast, cytosolic and mitochondrial fractions showed no or only slight change after the ischemia. TUNEL positive cells appeared at 48 h after the reperfusion, which was preceded by loss of bag-1 immunoreactivity. The present results suggest that bag-1 plays some roles in normal neuronal function, and its loss may be involved in neuronal cell death after ischemia.

Tamoxifen induces TGF-beta 1 activity and apoptosis of human MCF-7 breast cancer cells in vitro.

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-beta 1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-beta was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-beta.

Expression of inhibitor of apoptosis proteins (IAPs) in rat granulosa cells during ovarian follicular development and atresia.

The inhibitor of apoptosis proteins (IAPs) constitute a family of highly conserved apoptosis suppressor proteins that was originally identified in baculoviruses. Although IAP homologs have been recently identified and demonstrated to suppress apoptosis in mammalian cells, their expression and role during follicular development and atresia are unknown. The present study was conducted to address these questions. Using established in vivo models for the induction of follicular development and atresia in immature rats, it was possible to compare the immunolocalization of X-link inhibitor of apoptosis protein (Xiap) and human inhibitor of apoptosis protein-2 (Hiap-2), two members of the IAP family, at defined stages of follicular maturation and to relate the differences observed with those of follicular cell proliferation and apoptosis [as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL), respectively]. In addition, granulosa cell DNA and proteins were assessed for apoptotic fragmentation by 3'-end labeling/agarose gel electrophoresis (DNA ladder) and Hiap-2 and Xiap protein content by Western blot analysis, respectively. Hiap-2 and Xiap expression in both granulosa and theca cells increased with follicular maturation, reaching maximal levels at the antral stage of development. The immunoreactivity for PCNA, Xiap, and Hiap-2 decreased markedly in atretic (TUNEL-positive) follicles at the small to medium sized antral stage of development, suggesting follicular atresia may be associated with decreased granulosa cell IAP protein content and decreased proliferation. Atresia was also associated with a change in the intracellular distribution of IAPs in granulosa cells. Biochemical analysis of DNA fragmentation (DNA ladder) in granulosa cells from preantral and early antral follicles indicates extensive apoptosis that was associated with minimal IAP protein content. Gonadotropin treatment increased Hiap-2 and Xiap protein content and suppressed apoptosis in granulosa cells, resulting in the development of follicles to the antral and preovulatory stages. In addition, gonadotropin withdrawal induced apoptotic DNA fragmentation in granulosa cells in early antral and antral follicles, which is accompanied by a marked decrease in Hiap-2 and Xiap expression. These data suggest that IAPs may be involved in the suppression of granulosa cell apoptosis by gonadotropin in small to medium-sized antral follicles and play an important role in determining the fate of the cells, and thus also the eventual follicular destiny (atresia vs. ovulation).

Expression of Inhibitor of Apoptosis Proteins (IAPs) in Rat Granulosa Cells during Ovarian Follicular Development and Atresia

The inhibitor of apoptosis proteins (IAPs) constitute a family of highly conserved apoptosis suppressor proteins that was originally identified in baculoviruses. Although IAP homologs have been recently identified and demonstrated to suppress apoptosis in mammalian cells, their expression and role during follicular development and atresia are unknown. The present study was conducted to address these questions. Using established in vivo models for the induction of follicular development and atresia in immature rats, it was possible to compare the immunolocalization of X-link inhibitor of apoptosis protein (Xiap) and human inhibitor of apoptosis protein-2 (Hiap-2), two members of the IAP family, at defined stages of follicular maturation and to relate the differences observed with those of follicular cell proliferation and apoptosis [as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL), respectively]. In addition, granulosa cell DNA and proteins were assessed for apoptotic fragmentation by 3′-end labeling/agarose gel electrophoresis (DNA ladder) and Hiap-2 and Xiap protein content by Western blot analysis, respectively. Hiap-2 and Xiap expression in both granulosa and theca cells increased with follicular maturation, reaching maximal levels at the antral stage of development. The immunoreactivity for PCNA, Xiap, and Hiap-2 decreased markedly in atretic (TUNEL-positive) follicles at the small to medium sized antral stage of development, suggesting follicular atresia may be associated with decreased granulosa cell IAP protein content and decreased proliferation. Atresia was also associated with a change in the intracellular distribution of IAPs in granulosa cells. Biochemical analysis of DNA fragmentation (DNA ladder) in granulosa cells from preantral and early antral follicles indicates extensive apoptosis that was associated with minimal IAP protein content. Gonadotropin treatment increased Hiap-2 and Xiap protein content and suppressed apoptosis in granulosa cells, resulting in the development of follicles to the antral and preovulatory stages. In addition, gonadotropin withdrawal induced apoptotic DNA fragmentation in granulosa cells in early antral and antral follicles, which is accompanied by a marked decrease in Hiap-2 and Xiap expression. These data suggest that IAPs may be involved in the suppression of granulosa cell apoptosis by gonadotropin in small to medium-sized antral follicles and play an important role in determining the fate of the cells, and thus also the eventual follicular destiny (atresia vs. ovulation).