Development of a novel protocol for isolation and purification of human granulosa cells.

PURPOSE: To develop an optimal method of isolation and purification of human granulosa cells from ovarian follicular fluid. METHODS: Follicular fluid was collected from patients undergoing oocyte retrieval. A series of isolation and purification techniques was performed, involving density gradient centrifugation and use of different antibody-bead complexes. RESULTS: The highest percent yield of live purified granulosa cells came from density gradient centrifugation using sucrose polymer followed by positive selection of granulosa cells using primary antibody to MISRII and secondary antibody coupled to iron oxide beads. CONCLUSIONS: A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.

Steroid receptor coactivator-2 expression in brain and physical associations with steroid receptors.

Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ER(alpha) were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ER(alpha) bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ER(alpha) and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERbeta. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together, these findings reveal that SRC-2 is expressed in brain regions known to mediate a variety of steroid-dependent functions. Furthermore, SRC-2 is expressed in many ER(alpha) containing cells in the hypothalamus. Finally, SRC-2 from brain interacts with ER and PR in a subtype-specific manner, which may contribute to the functional differences of these steroid receptor subtypes in brain.

Oct-4A isoform is expressed in human cord blood-derived CD133 stem cells and differentiated progeny.

OBJECTIVES: This study aims to establish whether the pluripotent embryonic stem cell marker and nuclear transcription factor Oct-4A isoform is expressed in human umbilical cord blood CD133 stem cells (CD133 cells) and their differentiated progeny. MATERIALS AND METHODS: CD133 cells were examined for expression of the embryonic stem cell marker Oct-4A by reverse transcription-polymerase chain reaction using primers specific for the coding region of the Oct-4A isoform. Immunocytochemistry and flow cytometry were performed using an antibody raised to a peptide from the unique amino-terminal domain of the Oct-4A isoform, that does not exist in the Oct-4B isoform. Furthermore, specificity was confirmed by pre-adsorption of the antibody with the peptide immunogen. Differentiation was determined before and after expansion in culture, by flow cytometry for haematopoietic stem cell and differentiation markers. For many studies, after 7 days of culture CD133-positive and CD133-negative cells were separated by flow cytometry for additional analyses. Multilineage haematopoietic proliferative potential was determined using colony-forming assays. RESULTS: Freshly isolated CD133 cells expressed Oct-4A mRNA and protein. The cells proliferated rapidly in culture producing only a small proportion of CD133-positive cells and a much larger proportion of non-self-renewing CD133-negative cells. Proliferation was also associated with loss of other adult stem cell markers, gain of differentiated haematopoietic markers, and maintenance of potential to generate haematopoietic lineages. Oct-4A mRNA and protein were expressed throughout these changes. CONCLUSIONS: Oct-4A, which is associated with self-renewal in embryonic stem cells, neither defines nor confers self-renewal to CD133 stem cells.

Directed engineering of umbilical cord blood stem cells to produce C-peptide and insulin.

OBJECTIVES: In this study, we investigated the potential of umbilical cord blood stem cell lineages to produce C-peptide and insulin. MATERIALS AND METHODS: Lineage negative, CD133+ and CD34+ cells were analyzed by flow cytometry to assess expression of cell division antigens. These lineages were expanded in culture and subjected to an established protocol to differentiate mouse embryonic stem cells (ESCs) toward the pancreatic phenotype. Phase contrast and fluorescence immunocytochemistry were used to characterize differentiation markers with particular emphasis on insulin and C-peptide. RESULTS: All 3 lineages expressed SSEA-4, a marker previously reported to be restricted to the ESC compartment. Phase contrast microscopy showed all three lineages recapitulated the treatment-dependent morphological changes of ESCs as well as the temporally restricted expression of nestin and vimentin during differentiation. After engineering, each isolate contained both C-peptide and insulin, a result also obtained following a much shorter protocol for ESCs. CONCLUSIONS: Since C-peptide can only be derived from de novo synthesis and processing of pre-proinsulin mRNA and protein, we conclude that these results are the first demonstration that human umbilical cord blood-derived stem cells can be engineered to engage in de novo synthesis of insulin.

Production of stem cells with embryonic characteristics from human umbilical cord blood.

When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Inhibition of vascular endothelial growth factor (VEGF) does not affect early renal changes in a rat model of lean type 2 diabetes.

Type 2 diabetes is the most frequent cause of end-stage renal failure in many Western countries. Approximately 10-15 % of all type 2 diabetics are lean. Various growth factors and cytokines have been implicated in the pathophysiology of diabetic kidney disease, including vascular endothelial growth factor (VEGF). To elucidate a role for VEGF in the renal changes associated with type 2 diabetes, we examined the effect of a VEGF-antibody (ab) on early renal changes in the Goto-Kakizaki (GK) rat, a lean type 2 diabetes model. GK-rats were treated for 6 weeks with the VEGF-ab or with an isotype-matched irrelevant IgG. Wistar rats were used as non-diabetic controls. Placebo-treated GK-rats showed a pronounced increase in glomerular volume and urinary albumin excretion (UAE) and no change in the renal expression of endothelial nitric oxide synthase (eNOS) compared to placebo-treated non-diabetic controls. Kidney weight and creatinine clearance were no different between the groups. VEGF-ab treatment had no effect on glomerular volume, UAE, eNOS expression, body weight, blood glucose levels or food intake, but lowered serum insulin levels in non-diabetic and diabetic animals. We conclude that VEGF inhibition has minimal effects on early renal changes in GK-rats.

A murine vascular endothelial growth factor antibody inhibits in vivo growth of human Caki-I renal adenocarcinoma.

A number of studies have provided indirect evidence that angiogenesis is involved in tumour growth and metastasis formation of renal cell carcinoma (RCC). Nude mice bearing xenografts of human Caki-I RCC were treated i.p. for 3 weeks with a murine monoclonal antibody against vascular endothelial growth factor, VEGF (VEGF-ab). Tumour growth and mRNA expression of human and murine VEGF and murine VE-Cadherin in the tumours were measured. After 3 weeks of therapy, the tumour volume in the control nude mice was 548 +/- 98 mm3 compared to the tumours in the nude mice treated with VEGF-ab (122 +/- 24 mm3, p < 0.01). Treatment with VEGF-ab significantly reduced mRNA expression of murine VEGF-120 and murine VE-Cadherin (p < 0.05 and p < 0.01, respectively). The mRNA expression of human VEGF (hVEGF165, hVEGF189) and murine VEGF (mVEGF164) was unchanged due to antibody treatment. The mean percentage of apoptotic cells in tumours harvested from antibody-treated animals was significantly lower than in tumours from the control-treated animals (p < 0.02). These findings demonstrate for the first time that VEGF-ab significantly inhibit the growth of Caki-1 RCC in vivo.

Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.

BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells.

We have reported previously that among human prostate cancer cell lines LNCaP but not PC-3 cells undergo apoptosis after treatment with the protein kinase inhibitor staurosporine (STS). We have now further investigated this model to uncover the molecular mechanism causing resistance to STS-induced apoptosis in PC-3 cells. S-100 lysates of both cell lines showed biochemical changes typical of apoptosis after the addition of cytochrome c and dATP, suggesting that the postmitochondrial phase of apoptosis was intact. Upon addition of STS, the proapoptotic molecules Bax and Bad became predominantly mitochondrial in both cell lines. This, in turn, was followed by loss of mitochondrial transmembrane potential, translocation of cytochrome c to the cytosol, activation of caspase-9, -3, and -7, and cleavage of the apoptotic targets, DNA fragmentation factor and poly(ADP-ribose) polymerase, in LNCaP but not in PC-3 cells. Components of the mitochondrial permeability transition pore, adenine nucleotide transporter and voltage-dependent anion channel, were normally expressed in the correct subcellular fraction of both cell lines. Overexpression of the proapoptotic proteins Bax and Bad, fused to a green fluorescent protein but not of green fluorescent protein alone, induced apoptosis in >80% of PC-3 cells. These experiments suggested that a factor protecting the mitochondria of PC-3 cells mediates resistance to STS-induced apoptosis. A wide search among the antiapoptotic Bcl-2 family members was performed, and Bcl-X(L) was found to be overexpressed in PC-3 cells. Experiments down-regulating Bcl-X(L) expression by using the tyrosine kinase inhibitor genistein, sodium butyrate, or an antisense Bcl-X(L) oligonucleotide restored sensitivity to apoptosis in PC-3 cells. Thus, Bcl-X(L) overexpression is one of the mediators of resistance to STS-induced apoptosis in the prostate cancer cell line PC-3.

Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer.

Using adenoviral technology, we overexpressed the proapoptotic molecules pro-caspase-3, pro-caspase-7, and Bax to induce therapeutic apoptosis of prostate cancer cell lines growing in vitro and in vivo. Because overexpressed pro-caspase-3 did not undergo autocatalytic activation in any of the five prostate cancer cell lines evaluated, this strategy was unable to engage any component of the apoptotic pathway. Overexpressed pro-caspase-7 was proteolytically cleaved in LNCaP and LnCaP-Bcl-2 cells but not in PC-3, DU-145, or TsuPr(1) cells. Cleavage was associated with engagement of many components of the apoptotic pathway, including DEVDase activity, cleavage of intracellular caspase targets such as the DNA fragmentation factor and the proapoptotic Bid, release of cytochrome c from the mitochondria to the cytoplasm, and terminal deoxynucleotidyl transferase-mediated nick end labeling. No apoptosis was observed in the cells where caspase-7 did not undergo autocatalytic activation. Searching for an approach that would more reliably induce therapeutic apoptosis of prostate cancer cell lines, we used a binary adenoviral system to overexpress the proapoptotic molecule Bax. Bax was dramatically overexpressed and caused apoptosis of every cell line infected by engaging the mitochondrial pathway, including proteolytic cleavage and catalytic activation of the caspases, cleavage of caspase substrates, release of cytochrome c from the mitochondria, and DNA fragmentation. Furthermore, three injections of the Bax overexpression system into PC-3 cell tumors in nude mice in vivo caused a 25% regression in tumor size corresponding to a 90% reduction relative to continued tumor growth in animals that received injections with the control binary system expressing Lac-Z. These experiments show that adenovirus-mediated Bax overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo by activating the mitochondrial pathway of apoptosis. On the basis of these studies, we conclude that manipulation of Bax expression is an attractive new gene therapy approach for the treatment of prostate cancer.

Stroke and ischemia-reperfusion injury.

This annual meeting covered all aspects of the field of neuroscience. Stroke is one of the leading causes of morbidity, and this report will focus on the vast number of presentations on stroke and ischemia-reperfusion injury presented at the meeting.

3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9.

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.

Induction of apoptosis in BPH stromal cells by adenoviral-mediated overexpression of caspase-7.

PURPOSE: We hypothesized that expression/activity of critical components of the apoptotic pathway can be used to induce apoptosis of a human prostate cell line derived from benign prostatic hyperplasia (BPH) tissue. MATERIALS AND METHODS: We analyzed the apoptotic pathway in BPH cells treated with the powerful inducer of apoptosis, staurosporine (STS), and adenoviruses overexpressing caspase-3, -7, or the control gene lacZ. RESULTS: Twelve hours post-STS, most BPH cells were floating in the culture medium, TUNEL staining was widespread, and DEVDase activity (the catalytic activity of type II caspases) was increased. The pan-caspase inhibitor, Z-VAD-FMK, prevented STS-induced apoptosis. Based on these observations, we performed immunoblot analysis for the three known group II caspases (that is caspase-2, -3 and -7), but none of them was detected with three commercially available antibodies. Nevertheless, in view of the presence of increased DEVDase activity, we reasoned that a group II caspase must be a critical mediator of apoptosis in this model. If correct, we postulated that overexpression and activation of a type II caspase should cause apoptosis. To test this hypothesis, we coupled the cDNAs encoding caspase-3 and caspase-7 to adenoviral vectors and obtained constructs AvC3 and AvC7. Cells infected with AvC3 or AvC7 overexpressed the protein for caspase-3 or -7 within 24 to 48 hours. Caspase-3 overexpression did not cause apoptosis above that observed in cells receiving the control adenovirus expressing the lacZ cDNA (AvLac-Z). In contrast, caspase-7 overexpression induced massive apoptosis. BPH cells were then infected with increasing multiplicity of infection (MOI) of AvC7 and AvlacZ. A positive correlation was found between the amount of caspase-7 expressed and the level of DEVDase activity measured. AvC7 at MOIs of 25:1 and 50:1 induced apoptosis in about 50% of BPH cells at 72 hours post-infection. This effect was AvC7 specific, because the same MOIs of AvlacZ were not apoptogenic. CONCLUSIONS: Adenoviral-mediated overexpression of caspase-7 induces apoptosis of BPH-derived cells.

Heterogeneous apoptotic responses of prostate cancer cell lines identify an association between sensitivity to staurosporine-induced apoptosis, expression of Bcl-2 family members, and caspase activation.

BACKGROUND: The goal of this work was to identify mechanisms for the inability of metastatic prostate cancer cells to engage the apoptotic pathway following hormonal or cytotoxic therapy. METHODS: Genotypically diverse cell lines isolated from patients with metastatic disease were used. RESULTS: The LNCaP and TsuPr(1) lines exhibited quintessential apoptotic features in response to the pleiotropic apoptotic inducer staurosporine (STS): rapid cytochrome c translocation to the cytosol, proteolytic processing and catalytic activation of caspase-3 and -7, proteolytic inactivation of the death substrates DNA fragmentation factor (DFF) and poly-ADP-ribose polymerase (PARP), and TUNEL-positive polyfragmented nuclei. In contrast, DU-145 and PC-3 cells exhibited few, if any, of these features, while appearing necrotic by confocal microscopy. The presence of caspase-3 and -7 without proteolytic processing suggested that the apoptotic blockade was upstream of executioner caspases in these resistant cell lines. To identify the locus of this block, Western blot analysis of cytochrome c subcellular localization and of pro- and antiapoptotic Bcl-2 family members was performed, and suggested that heterogeneous expression of these proteins might be the underlying mechanism for apoptotic resistance to STS in these cell lines. Thus, the absence of the proapoptotic Bax in DU-145 cells indicated a mechanism for apoptotic resistance of these cells. Similarly, decreased Bax expression during STS treatment, coupled with overexpression of the antiapoptotic Bcl-x(L) and inability to translocate cytochrome c to the cytosol, provided a mechanism for the insensitivity of PC-3 cells. CONCLUSIONS: These observations suggest that activation of the apoptotic machinery in metastatic prostate cancer cell lines may be determined by expression levels of Bcl-2 family members, by the ability of cytochrome c to translocate to the cytosol, and by the ability of the caspase pathway to react in response to activation of the mitochondrial phase.

Nuclear factor-kappaB and cell death after experimental intracerebral hemorrhage in rats.

BACKGROUND AND PURPOSE: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-kappaB in relation to cell death surrounding intracerebral hemorrhage (ICH). METHODS: Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-kappaB protein, Western blot was performed on brain extract. We determined NF-kappaB activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-kappaB. DNA fragmentation was detected with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining. RESULTS: Western blot analysis of the NF-kappaB p65 subunit showed that there was no difference in p65 protein levels in the control, 2-hour, 8-hour, or 24-hour groups. However, ipsilateral perilesional samples from the 4-day group revealed a 1.8- to 2.5-fold increase compared with the contralateral hemisphere. Western blotting showed no differences in the inhibitor of NF-kappaB, IkappaBalpha, in any group. EMSA showed 1.3-, 2.1-, and 3.6-fold increased NF-kappaB activation in the ipsilateral striatum from the 8-hour, 24-hour, and 4-day groups, respectively, compared with the contralateral hemisphere. Immunohistochemistry, in which an activation-dependent anti-NF-kappaB antibody was used, demonstrated perivascular NF-kappaB activation as early as 2 hours after ICH with more generalized activation at 8 hours, in agreement with the EMSA results. NF-kappaB activation colocalized to cells containing fragmented DNA measured by TUNEL. CONCLUSIONS: The present study suggests a relationship between NF-kappaB and the pathobiology of perilesional cell death after ICH.

Signaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer.

We studied the molecular mechanisms of apoptosis in the prostate cancer cell line LNCaP and whether overexpression of caspase activity could force this cell line to undergo apoptosis. The inhibitor of phosphomevalonate decarboxylase, sodium phenylacetate, and the protein kinase inhibitor staurosporine induced (a) release of cytochrome c from the mitochondria to the cytosol; (b) reduction in mitochondrial transmembrane potential; (c) proteolytic processing of caspase-3 and -7 but not -2; (d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. The panspecific inhibitor of caspase activation N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of these events except release of mitochondrial cytochrome c into the cytosol. None of these apoptotic signaling events were elicited by staurosporine or sodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpress the oncoprotein Bcl-2. Because caspase-7 is activated in every model of apoptosis that we have characterized thus far, we wished to learn whether overexpression of this protease could directly cause apoptosis of LNCaP cells. By using a replication-defective adenovirus, overexpression of caspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompanied by induction of cleavage of the DEVD substrate and TUNEL. These studies have demonstrated that caspase-7 and -3 are critical mediators of apoptosis in LNCaP cells. Caspase-7 was proteolytically activated in every model of apoptosis that we have developed, and the overexpression of it induced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for the treatment of prostate cancer.

Caspases in apoptotic death.

Caspases are the central mediators of normal and pathological apoptotic death. They are cysteine proteases that cleave after aspartic acid. The inactive pro-enzymes are proteolytically processed and activated through mechanisms of recruitment to signalling complexes, upstream activator caspases or autoactivation. Many amplifying cascades exist in caspase activation pathways that are evolutionarily conserved. The human caspase family contains at least 10 members which form 3 groups based on substrate specificity, proteolytic targets and functional actions. The crystal structure has been solved for members from two of these groups with inhibitory peptides bound in the active site. These structures have allowed tremendous insight into mechanisms of catalysis, substrate binding and substrate specificity. Enlightened knowledge of enzyme-substrate interactions has led to the design of many inhibitors that are active in animal models of caspase-mediated cell death. Testing in animal models should lead to drugs for therapeutic intervention in the many human diseases characterised by excessive apoptotic cell death.

Alzheimer's disease and stroke.

This report focuses on the two most common neurological diseases in man: Alzheimer's disease (AD) and stroke. One common feature of both of these diseases is the death of cells, particularly neurons. Since a typical mechanism of cell death is apoptosis, this will be an additional focal point in this report.

Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP.

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.