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Differentiation-induction therapy is an attractive approach in leukemia treatment. It has been suggested that the accumulation of intracellular reactive oxygen species (ROS) is involved in megakaryocytic differentiation induced by phorbol 12-myristate 13-acetate (PMA) in the K562 leukemia cell line. Therefore, a ROS-inducible technique could be a powerful method of differentiation induction. Accordingly, we hypothesized that ionizing radiation contributes to the acceleration of megakaryocytic differentiation through the accumulation of intracellular ROS in leukemia cells. In the present study, ionizing radiation was shown to promote PMA-induced megakaryocytic differentiation. Cells with high CD41 expression sustained intracellular ROS levels effectively. The enhancement of differentiation by ionizing radiation was found to be regulated through the mitogen-activated protein kinase (MAPK) pathway, involving both extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 MAPK. Ionizing radiation also controlled mRNA expression of the oxidative stress response gene heme oxygenase-1 (HO1). Consequently, we concluded that intracellular ROS, increased by ionizing radiation, modulate megakaryocytic differentiation downstream of the MAPK pathway.

Megakaryocytes are generated by the differentiation of megakaryocytic progenitors; however, little information has been reported regarding how ionizing radiation affects the differentiation pathway and cellular responses. Human leukemia K562 cells have been used as a model to study megakaryocytic differentiation. In the present study, to investigate the effects of radiation on phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation of K562 cells, the cellular processes responsible for the expression of CD41 antigen (GPIIb/IIIa), which is reported to be expressed early in megakaryocyte maturation, were analyzed. The expression of CD41 antigens was significantly increased 72 h after treatment with both 4 Gy X-irradiation and PMA. In this fraction, two populations, CD41(low) and CD41(high) cells, were detected by flow cytometry. The CD41(high) cells sustained intracellular ROS at the initial level for up to 72 h, but CD41(low) cells had reduced ROS by 48 h. The maximum suppressive effect on CD41 expression was observed when N-acetyl cysteine, which is known to act as a ROS scavenger, was administered 48 h after PMA stimulation. When K562 cells were pretreated with mitogen-activated protein kinase (MAPK) pathway inhibitors, an ERK1/2 inhibitor and a p38 MAPK inhibitor, followed by X-irradiation and PMA stimulation, the reactivity profiles of both inhibitors showed the involvement of MAPK pathway. There is a possibility that the K562 cell population contains at least two types of radiosensitive megakaryocytic progenitors with respect to ROS production mechanisms, and intracellular ROS levels determine the extent of CD41 expression.

Oxidative stress was induced in 12-week-old offspring of protein-restricted (9% protein) and control (20% protein) protein-restricted stroke-prone spontaneously hypertensive rats (SHRSP) by administering phorbol 12-myristate 13-acetate (PMA) for 4 weeks to determine the effects of oxidative stress on the vascular function of the SHRSP offspring. There was no significant difference in the blood pressure of offspring of the protein-restricted dams and control dams. The plasma diacron-reactive oxygen metabolite (dROM) level at 16 weeks of age was significantly higher in offspring of the protein-restricted dams, whereas the anti-oxidative enzyme activity was similar in both groups. Acetylcholine (Ach)-induced relaxation was significantly reduced in offspring of the protein-restricted dams. The expression of endothelial nitric oxide synthase (eNOS) was lower and the expression of soluble guanylic acid cyclase (sGC) was higher in offspring of the protein-restricted dams. These results indicate that SHRSP offspring of the protein-restricted dams were sensitive to oxidative stress, and displayed the vascular dysfunction.

Oocyte competence is the ability of the oocyte to complete maturation, undergo successful fertilization, and reach the blastocyst stage. Cumulus cells are indispensable for this process. Their removal significantly affects the blastocyst rates. Moreover, the properties and functions of cumulus cells are regulated by the oocyte. They also reflect the oocyte's degree of maturation. Our study was aimed at identifying markers of oocyte competence that are expressed in bovine cumulus cells. In a previous study in our laboratory, the blastocyst yield following FSH or phorbol myristate acetate (PMA) treatment was 45%%. Therefore, we tested four sets of conditions during the first 6 h of in vitro maturation (IVM): FSH (0.1 microg/ml), PMA (0.1 microM), FSH ++ PMA, and negative control. Extracts from each IVM treatment were hybridized against the same negative control on a microarray containing a partial library of differentially expressed transcripts in the cumulus of competent oocytes collected at 6 h after LH in vivo. Common positive clones between diffrentially treated cells were selected, and 15 candidates were validated by real-time PCR. Based on this, the main candidates expressed in cumulus cells and that could be valuable and indirect markers of oocyte competence are hyaluronan synthase 2 (HAS2), inhibin betaA (INHBA), epidermal growth factor receptor (EGFR), gremlin 1 (GREM1), betacellulin (BTC), CD44, tumor necrosis factor-induced protein 6 (TNFAIP6), and prostaglandin-endoperoxide synthase 2 (PTGS2). These biomarkers could be potential candidates to predict oocyte competence and to select higher-quality embryos for transfer. Additionally, these indirect predictors of oocyte competence and follicular health could improve our knowledge of gene expression patterns in the cumulus and yield insights into the molecular pathways controlling oocyte competence.

In essential hypertension (EHT) the presence of a metabolic syndrome increases the risk of cardiovascular disease. A cell membrane abnormality is implicated but its role in cardiovascular disease is unclear. Neutrophil accumulation, which occurs by beta2-integrin (CD11b/CD18) expression, followed by release of proinflammatory factors from primary vesicles is an important factor in vascular damage. CD11b and CD69 expression on neutrophils from normal controls and EHT patients was determined by fluorescence-activated cell scanning. Neutrophils were activated with phorbol myristate acetate (PMA). Protein kinase C (PKC) and calpain were inhibited with bisindolylmaleimide and E64d, respectively. In EHT patients CD11b was not increased on neutrophils at rest. However, EHT neutrophils more readily expressed CD11b on incubation in phosphate-buffered saline and more cells went on to exocytose primary granules indicated by expression of CD69. Stimulation with PMA caused more rapid activation in EHT neutrophils with expression of CD11b, followed rapidly by exocytosis of primary granules. Bisindolylmaleimide slowed but did not prevent CD11b expression, which, together with primary granule exocytosis, continued to be faster in EHT neutrophils. E64d also slowed but did not prevent either CD11b expression or primary granule exocytosis, but this inhibitor did abolish the difference between NC and EHT neutrophils. The membrane abnormality in EHT may contribute to cardiovascular risk by increasing the rate of vesicle fusion with the cell membrane to increase neutrophil accumulation and release of inflammatory agents at sites of vascular damage. Calpain activation may be the rate-limiting component that is abnormal.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play very important roles in extracellular matrix (ECM) remodeling in ovarian follicle growth and ovulation. Equine follicles are embedded in cortex that is at the center of the ovary, and they must expand/emigrate to the fossa, the only site in the ovary for ovulation. Therefore, equine ovarian stromal cells (EOSC) are probably involved in ECM remodeling during follicle growth. This study examined whether cultured EOSC synthesize gelatinases and TIMPs, molecules essential for ECM remodeling in other systems. Results showed that cultured EOSC (passage 3-8) had a fibroblast-like morphology and were positive for alpha-smooth muscle actin and type I procollagen by immunostaining. Gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1, and TIMP-2 were present in EOSC-conditioned medium, and TIMP-3 in ECM of EOSC. Transforming growth factor beta significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium from EOSC (p < 0.05). Phorbol 12-myristate 13-acetate also significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium and of TIMP-3 in ECM (p < 0.05). Our results suggest that EOSC produce important components of the ECM remodeling machinery and, therefore, may play a role in the ECM remodeling during follicle growth in this species.

Expression of the POU domain protein Tst-1/Oct6 during development of glia and neurons is subject to a tight multifactorial control. Here we show that 17beta-estradiol increases the level of endogenous Tst-1/Oct6 in glial cells. This effect was mediated at the level of gene expression by an enhancer present in the 5' flanking region of the mouse gene for Tst-1/Oct6, approximately 5 kb upstream of the transcriptional start site. The enhancer contained as the functional element a sequence motif that closely resembled a classical estrogen response element. It consisted of an imperfect palindrome with a spacing of 3 bp, and was bound in vitro by activated estrogen receptor. Furthermore, this element was able to confer estrogen responsiveness when introduced into a heterologous promoter. In the Tst-1/Oct6 gene enhancer, a TPA response element was found in close proximity to the estrogen receptor binding site. As a consequence, TPA and estrogen activated transcription of the Tst-1/Oct6 gene in a synergistic manner.

The aims of these studies were to examine the effects of FCC5 (2-carboxamidino-1,2,3,4,10,14b-hexahydrodibenzo (c,f) pyrazino (1,2,-a) azepine HCl), an analogue of mianserin, on immediate type hypersensitivity reactions in-vitro. The actions of FCC5 were examined on the Schultz-Dale reaction of guinea-pig ileum and on histamine and leukotriene release from human- and guinea-pig-sensitized lung fragments. FCC5 (applied topically) was assessed for anti-inflammatory activity in-vivo against phorbol-12-myristate-13-acetate (PMA)-induced oedema in the mouse ear. FCC5 (IC50 = 0.17 microM) was a potent inhibitor of the Schultz-Dale reaction in-vitro, as assessed by a concentration-dependent attenuation of egg albumin-induced contractions of sensitized guinea-pig isolated ileum. Using human and guinea-pig isolated sensitized lung fragments, FCC5 (1-100 microM) attenuated antigen-induced release of sulphidopeptidoleukotrienes and histamine. FCC5 (50 micrograms topically) resembled mianserin and indomethacin in attenuating PMA-induced mouse ear inflammation. These properties together with previously published evidence of long lasting antihistamine properties in-vivo, suggest that FCC5 has therapeutic potential as an anti-allergic agent, especially in pathological conditions where an inflammatory component is present.

Experiment I was designed to determine the optimal dose of phorbol 12-myristate 13-acetate (PMA) that inhibited progesterone production when infused into the ovarian artery. The most efficacious dose of PMA was 2 mumol. Experiment II was designed to determine whether activation of protein kinase C (PKC) inhibited progesterone production without initiating luteolysis. Ewes received ovarian arterial infusions of 4 alpha-phorbol (2 mumol, n = 4), PMA (2 mumol, n = 8), or prostaglandin F2 alpha (PGF2 alpha; 1 mumol, n = 5). Concentrations of progesterone in serum decreased by 3 h in PMA-treated ewes and by 5 h in PGF2 alpha treated ewes (p < 0.05). By 48 h, serum levels of progesterone in PMA-treated ewes had returned to control values; but in PGF2 alpha-treated ewes they remained low for the duration of the experiment. Luteal weights and progesterone contents at 48 h were similar in 4 alpha-phorbol- and PMA-treated ewes but were decreased in PGF2 alpha-treated ewes (p < 0.05). Experiment III was designed to determine whether PGF2 alpha or PKC activation induced oligonucleosome formation or influenced mRNA levels for cytochrome P450sec or 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD). Ewes received treatments as in experiment II, and CL were collected at 3, 12, or 24 h (n = 3-4 per group). Luteal weights were decreased (p < 0.05) and oligonucleosome formation was increased (p < 0.05) in PGF2 alpha-treated ewes compared to controls or to PMA-treated ewes by 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Our previous study provided a novel assay system utilizing devitalized bone slices for study of the differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts among calvarial cells of mouse embryos. Using this assay system, we examined the effect of phorbol myristate acetate (PMA) on osteoclast formation as assessed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells and bone resorption lacunae. PMA alone was directly unable to induce the appearance of TRAP-positive cells and bone resorption lacunae of calvarial bone cells of mouse embryos. However, PMA markedly stimulated increases in the number of TRAP-positive cells and area of the resorption lacunae of the calvarial cells when the bone cells were primed by 1 alpha,25-(OH)2D3. This stimulatory effect of PMA was dose dependent. H-7, having relatively high affinity for protein kinase C, strongly inhibited in a dose-dependent fashion the stimulatory effect of PMA on the bone resorption of the hormone-primed calvarial cells. We also examined the involvement of prostaglandin in this stimulatory effect of PMA. Indomethacin, a cyclooxygenase inhibitor, markedly abolished the stimulatory effect of PMA on the bone resorption of the calvarial cells. PMA stimulated prostaglandin E2 (PGE2) production by the calvarial cells primed with 1 alpha,25-(OH)2D3 in a dose-dependent fashion. However, the PMA stimulation of the PGE2 production was significantly inhibited by H-7 and also by indomethacin. Furthermore, we observed that the addition of PGE2 to the calvarial cells primed with 1 alpha,25-(OH)2D3 for 1 or 3 days resulted in an increased number of TRAP-positive cells and increased bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (PKC), and the PKC inhibitor staurosporine on GnRH secretion and mRNA levels were studied in GT1-7 hypothalamic neuronal cells. Dose-response and time-course studies revealed that TPA (10(-8) M) acutely increased GnRH secretion 3-fold at 3-6 h, which then declined to baseline at 24 h, while it progressively decreased GnRH mRNA levels by 50% and 70% at 6 and 24 h, respectively. To ensure that these effects were due to activation and not down-regulation of PKC, cells were treated for 30 min with TPA (10(-8) M). This brief exposure to TPA also resulted in a decrease (60%) in GnRH mRNA levels at 6 h, with a 1.5- to 2-fold increase in GnRH secretion compared to control values, suggesting that activation of PKC decreases the pretranslational expression of GnRH while increasing GnRH secretion. Additional studies measured PKC activity and documented a shift from a cytosolic to a membrane fraction after incubation with TPA, again supporting PKC activation. Exposure of GT1-7 cells to staurosporine (10(-8) M), a PKC inhibitor, resulted in no change in the level of GnRH mRNA or secretion at 6 h. However, incubation with both TPA and staurosporine prevented the decrease in GnRH mRNA levels and partially blocked the increase in GnRH secretion induced by TPA. We conclude that TPA, by activating the PKC pathway, acutely increases GnRH secretion, but dramatically decreases GnRH gene expression. The exact mechanism of these divergent effects on the synthesis and secretion of GnRH remain to be elucidated.

Intralesional administration of emulsified croton oil into established syngeneic transplants of murine firosarcoma no. 1023 caused complete regression of the injected tumors in C3H mice without recurrence during the period of observation. In Sewall Wright strain 2 guinea pigs, in contrast to BCG cell wall vaccine which eradicated regional lymph node metastasis as well as dermal transplants, croton oil treatment only delayed the development of metastatic disease despite the fact that the injected skin tumors did not recur. 12-O-Tetradecanoylphorbol 13-acetate (TPA), the active principle of croton oil, incorporated in mineral oil droplets in aqueous suspension, caused regression of murine tumors when injected intralesionally. Aqueous suspensions of TPA failed to eliminate the tumors. Our results suggest that tumor regression induced by croton oil of TPA emulsions was due to indiscriminate destruction of the injected tissue.

The value of the SENCAR mouse for testing tumorigenic properties of complex mixtures on mouse skin was studied. Seven complex mixtures were obtained as dichloromethane extracts of collected particulate emissions from three diesel-fueled automobiles, a heavy-duty diesel engine, a nonleaded gasoline-fueled automobile, a coke oven battery, and a roofing tar pot. These emissions were applied topically at multiple doses to both male and female SENCAR mice that were subsequently promoted with 12-O-tetradecanoylphorbol 13-acetate. Two statistical analyses were applied to the data to rank the samples and to provide 95% confidence intervals. One analysis used tumor multiplicity data, applied them to a nonlinear Poisson model, and the second analysis used tumor incidence data and applied them to a log-probit model. Both analyses ranked the complex mixtures in similar order. Benzo[a]pyrene content alone could not account for all the tumorigenic activity in each complex mixture, indicating that other components also contribute to the overall tumorigenic activity.

Guinea-pig ears were treated topically with 20 nmol of 12-O-tetradecanoyl phorbol-13-acetate, or deprived of their horny layer by nine successive strippings. At different times after the treatment, the animals were sacrificed, the epidermis removed from the ears, and the RNA from the epidermis purified and analysed by dot-blot hybridization in order to assess and determine the amount of RNA able to hybridize to each one of the 18 onc-gene DNA probes. The following probes were used: v-src, v-fps, v-yes, v-ros, v-myc, c-myc, v-erb AB, v-myb, v-mos, v-Ha-ras, v-Ki-ras, v-abl, v-fos, v-fes, v-fms, c-sis, B-lym, v-raf. At 0 h, expression of B-lym and of myc and fos is seen. ErbAB mRNA is detected between 10 min and 4 h after stripping, as well as after TPA application. B-lym mRNA is detected for up to 36 h after stripping and for up to 8 h after TPA application. C-myc mRNa is detected for up to 36 h after tape stripping, but only for the first hour after TPA application. RNA complementary to the other onc probes was not detected, and synthesis of RNA complementary to an actin DNA probe was observed for 8 h after TPA application.

Dose-response relationships on the abilities of several phorbol ester tumor promoters to promote skin tumors after 7,12-dimethylbenz[a]anthracene initiation and to bring about edema, inflammation, and epidermal hyperplasia were determined in female Charles River CD-1 mice. The promoting ability of the potent synthetic promoter, phorbol-12,13-dioctanoate (PdiC8), was determined over a dose range of 0.1-10 mug/application. Administration of PdiC8 two times weekly at dosages of 4, 6, 8, and 10 mug gave little variation in tumor response. A dose-dependent tumor response occurred at doses of 1-4 mug PdiC8. Only 1 papilloma was observed when PdiC8 was given twice weekly at a dose of 0.1 or 0.5 mug. A similar dose-response relation was observed for the ability of PdiC8 to stimulate epidermal hyperplasia. Investigations of other phorbol esters revealed an excellent correlation between their promoting ability and their ability to induce epidermal hyperplasia; however, that was not the case for compounds outside the phorbol ester series (i.e., acetic acid, cantharidin, and ethylphenylpropiolate).