Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer.

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia.

BACKGROUND AND PURPOSE: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. EXPERIMENTAL APPROACH: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. KEY RESULTS: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

A frequency stabilization technique for diode lasers based on frequency-shifted beams from an acousto-optic modulator.

We present a simple method for diode laser frequency stabilization that makes use of a Doppler-broadened vapor cell absorption signals of two frequency-shifted laser beams. Using second-order-diffracted, double-passed beams from an acousto-optic modulator, we achieve a frequency separation roughly equal to the Doppler half width. The differential transmission signals of the two beams provide an error signal with a very large linear feature, allowing frequency stabilization over a range of greater than 1 GHz by means of standard proportional-integral-derivative servo feedback to the piezoelectric control of the grating in our external cavity diode laser. We have applied this technique to two different diode laser systems, one used to lock to the 410 nm E1 transition in indium and another for locking to the M1/E2 transition in thallium at 1283 nm. In both cases the technique reduces frequency fluctuation to roughly 1 MHz over time scales from 10(-3) to 10(2) s.

In vivo modulation of androgen receptor by androgens.

AIM: To study the effect of androgen and antiandrogen on the level of androgen receptor (AR) mRNA. METHODS: The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated. RESULTS: Androgenic deprivation produced by castration decreased the weight of the prostate and increased the levels of AR mRNA. Treatment of the castrated rats with testostrone increased the weight of prostate and decreased the levels of AR mRNA. Treatment of normal rats with flutamide decreased the weight of the gland and increased the levels of AR mRNA. CONCLUSION: Androgens produce proliferative effect on the prostate and negatively regulate the AR transcription.

Targeting of protein kinase C delta to mitochondria in the oxidative stress response.

The cellular response to oxidative stress includes the release of mitochondrial cytochrome c and the induction of apoptosis. Here we show that treatment of diverse cells with hydrogen peroxide (H2O2) induces the targeting of protein kinase C delta (PKCdelta) to mitochondria. The results demonstrate that H2O2-induced activation of PKCdelta is necessary for translocation of PKCdelta from the cytoplasm to the mitochondria. The results also show that mitochondrial targeting of PKCdelta is associated with the loss of mitochondrial transmembrane potential and release of cytochrome c. The functional importance of this event is also supported by the demonstration that H2O2-induced apoptosis is blocked by the inhibition of PKCdelta activation and translocation to mitochondria. These findings indicate that mitochondrial targeting of PKCdelta is required, at least in part, for the apoptotic response of cells to oxidative stress.

Observations on EGFR gene amplification and polymorphism in prostatic diseases.

This study was designed to determine the amplification and polymorphism of epidermal growth factor receptor (EGFR) gene in prostatic diseases like benign hypertrophy (BPH) and carcinoma (CaP). The EGFR gene was found to be amplified in grade IV BPH as compared to grade II BPH. Digestion of genomic DNA with MspI and HpaII revealed the presence of a 5kb band following southern blot analysis. This 5kb band was present in all the CaP cases and in one out of three BPH cases. It is possible that such a polymorphism is associated with the type or extent of tumor progression.

Mitochondrial translocation of protein kinase C delta in phorbol ester-induced cytochrome c release and apoptosis.

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) delta from the cytoplasm to mitochondria. The results also show that translocation of PKCdelta results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCdelta translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCdelta to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation.

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.

Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase.

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70(S6k)) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation.

Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism.

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.

Bcl-xL blocks activation of related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2 and stress-activated protein kinase/c-Jun N-terminal protein kinase in the cellular response to methylmethane sulfonate.

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.

Detection of receptor transcripts for androgen, epidermal growth factor and basic fibroblast growth factor in human prostate postmortem.

RNA isolated from frozen human postmortem prostate tissue was evaluated for its utility in molecular biological studies based on RNA analysis. Our results on slot-blot analysis show the presence of receptor transcripts for androgen, epidermal growth factor and basic fibroblast growth factor in postmortem prostate tissue obtained 20-120 hrs after death. However, the RNA in these samples was found to be degraded as revealed by the absence of ribosomal bands on gel electrophoresis. AR mRNA was found to be present in one of the five samples when analysed by northern blotting.

Comparative analysis of epidermal growth factor receptor mRNA levels in normal, benign hyperplastic and carcinomatous prostate.

Epidermal growth factor receptor (EGFR), a mediator of mitogenic activity of epidermal growth factor and transforming growth factor-alpha, has been shown to be associated with tumour progression. We have detected a level of EGFR transcript in normal, hyperplastic (BPH) and carcinomatous (CaP) prostate tissues by cytoplasmic dot hybridization. Our results show that the majority of CaP cases (62% of untreated cases and 71% of treated cases) were positive for EGFR mRNA while about 50% of normal and BPH cases were positive for EGFR mRNA. The level of EGFR transcript was significantly higher in the untreated CaP group as compared to the normal group (P < 0.05), while the difference in the normal and BPH groups was not statistically significant.

Anti-steroidogenic activity of the petroleum ether extract and fraction 5 (fatty acids) of carrot (Daucus carota L.) seeds in mouse ovary.

The petroleum ether extract and fraction 5 (fatty acids) of carrot seeds arrested the normal estrus cycle of adult mouse and reduced the weight of ovaries significantly. The cholesterol and ascorbic acid content in ovaries were significantly elevated due to the treatment with extract and fraction 5 (fatty acids) of carrot seeds. The significant inhibition of delta 5,3-beta-hydroxy steroid dehydrogenase and glucose-6-phosphate dehydrogenase, the two key enzymes involved in ovarian steroidogenesis, were also observed in mouse ovaries after 15 days of treatment. Results of this study revealed that the fraction 5 (fatty acids) present in carrot seeds acts as an antisteroidogenic agent.

Androgen deprivation causes up-regulation of androgen receptor transcript in the rat prostate.

We have studied the effect of androgenic deprivation on the level of androgen receptor transcript in the rat ventral prostate. The rats were treated with estradiol benzoate, flutamide and [D Trp6, des Gly10]gonadotropin releasing hormone (GnRH) for different time periods. These treatments produced a significant decrease in the weight of prostate. Total RNA isolated from the ventral prostates was hybridized with the cDNA probe for androgen receptor. Densitometric analysis of the autoradiographic signal revealed a rise in the level of androgen receptor RNA following treatment of rats with estradiol benzoate and flutamide. Treatment of rats with [D Trp6, des Gly10] GnRH brought about a transient rise in the level of androgen receptor RNA. Thus, our results indicate that androgenic deprivation up-regulates the level of androgen receptor transcript.

Androgen receptor transcript level in benign hypertrophy and carcinoma of the human prostate.

OBJECTIVE: Current reports suggest the presence of androgen receptor (AR) defects in prostate cancer. We have detected the AR mRNA in normal, benign hypertrophied (BPH) and carcinomatous (CaP) prostate tissues and evaluated the difference in the level of AR transcript in these groups. MATERIALS AND METHODS: Cytoplasmic dot hybridization assay was used to measure the levels of AR mRNA in 21 normal, 45 BPH and 30 CaP specimens. Tissue samples of 14 CaP patients receiving endocrine treatment were also analyzed. Blots were autoradiographed and the intensity of the signal was measured by densitometry. RESULTS AND CONCLUSIONS: The majority of cases in all the 3 groups were positive for the AR mRNA. All the patients who had received endocrine treatment were also positive for the AR mRNA. The level of the AR mRNA was significantly higher in BPH and treated CaP cases as compared to normal cases (p < 0.05). Among the CaP cases the level of AR transcript was significantly higher in the treated group as compared to the untreated group.

Androgen receptor mRNA detection in the human foetal prostate.

In order to get an insight into androgen-mediated differentiation and development of the prostate, we detected the androgen receptor (AR) mRNA in the urogenital sinus of human foetuses at 12 and 16 weeks of gestation. Cytoplasmic dot hybridization using radiolabelled cDNA probe for human androgen receptor (AR) was performed. The AR mRNA could be detected at 12 weeks of gestation and its level was higher at 16 weeks of gestation.

Cardiovascular and respiratory changes following exposure to a synthetic toxin of Ptychodiscus brevis.

The present study demonstrated cardiorespiratory effects of a synthetic phosphorus-containing ichthyotoxic metabolite elaborated by the marine dinoflagellate Ptychodiscus brevis in anaesthetised cats. The metabolite at a dose of 0.25-1.5 mg/kg i.v., resulted in a dose-dependent fall in blood pressure and such vasodepressor effect was associated with bradycardia. There is initial respiratory apnoea followed by increased rate and depth of respiration (hyperapnoea) following the administration of the toxin. The hypotensive response was accompanied by a decrease in aortic baroreceptor activity. The ECG showed atrioventricular conduction block, arrhythmia and depression of S-T segment and T wave which indicated coronary insufficiency. Vasodepressive property of the toxin is presumably muscarinic in nature as atropine counteracted the vasodepression.

Prostate gland: structure, functions and regulation.

The prostate gland plays an important role in male reproduction. It secretes enzymes, lipids, amines and metal ions essential for the normal function of spermatozoa. Development, differentiation and maintenance of the prostate gland depend on steroid and peptide hormones. Beside hormones growth factors also regulate the prostate gland. This review will focus on the structure, functions and mode of regulation of the prostate gland.