Propidium Iodide-stained Nuclei Research Articles

Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

Estimation of the relative DNA content in species of the genus Spiraea, sections Chamaedryon and Glomerati by flow cytometry

The relative DNA content was studied in seven species of the genus Spiraea L., section Chamaedryon Ser., and in two species, section Glomerati Nakai, from 28 natural populations growing in Asian Russia. The cell nuclei were isolated from a leaf tissue. The relative intensity of fluorescence was measured using flow cytometry of propidium iodide-stained nuclei. The analysis was performed using a CyFlowSpace device (Germany, Sysmex Partec) with a laser radiation source of 532 nm. Fresh leaves of Solanum lycopersicum cv. ‘Stupice’ were used as an internal standard. Data on the relative DNA content are presented for the first time for S. flexuosa Fisch ex Cambess. (0.42–0.47 pg), S. ussuriensis Pojark. (0.49–0.52; 0.85 pg), S. alpina-Pall. (0.49–0.51 pg), S. media Schmidt. (0.45; 0.98–1.01 pg), S. trilobata L. (0.46 pg), S. hypericifolia L. (0.49–0.52 pg) and S. aquilegifolia Pall. (0.48–0.51 pg). Mesophytic species of the genus Spiraea growing in the forest zone (S. chamaedryfolia L. and S. media) exhibit a 2-fold higher relative DNA content (C-value) compared to xerophytic species (S. hypericifolia, S. crenata L., S. aquilegifolia and S. trilobata) and species confined only to mountain areas (S. alpinа, S. trilobata and S. aquilegifolia).

Tylophorine reduces protein biosynthesis and rapidly decreases cyclin D1, inhibiting vascular smooth muscle cell proliferation in vitro and in organ culture

BackgroundTylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. Hypothesis/PurposeOur goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. Study design and methodsTYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. ResultsTYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. ConclusionThis unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.

Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae)

Schoenus (Cyperaceae) has holocentric chromosomes. Chromosome numbers were counted and nuclear DNA amounts were measured for all the New Zealand species of the genus. Chromosome numbers ranged from 2n = 8 to c .2 n = 90. Two chromosome races, with 2n = 28 and 2n = 56, were found in S. pauciflorus. Flow cytometry using propidium iodide-stained nuclei was used to measure genome size. A 14.8-fold variation in 2C DNA content was found, with values ranging from 1.33 to 19.71 pg/2C nucleus. Phylogenetic trees based on sequence variation in the internal transcribed spacer (ITS) region of the 45S ribosomal DNA locus were constructed using several phylogenetic models to reveal possible evolutionary relationships among the New Zealand Schoenus spp. and a sample of Australian Schoenus spp. Analysis revealed heterogeneity of chromosome number, size and DNA C value within clades. Meiosis in four species showed only bivalent formation at metaphase I. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 555–564. ADDITIONAL KEYWORDS: flow cytometry – holocentric chromosomes – ITS phylogeny.

Estimation of the Glomus intraradices nuclear DNA content

Estimation of the <i>Glomus intraradices</i> nuclear DNA content

Matrigel-Induced Tubular Morphogenesis of Human Eccrine Sweat Gland Epithelial Cells

Human eccrine sweat glands are tubule-structured glands of the skin that are vital in thermoregulation, secretion, and excretion of water and electrolytes. A study of tubular morphogenesis in vitro would facilitate the development of a tissue engineering model for eccrine sweat glands and other tubule-structured glands. Matrigel, a basement membrane matrix, has been shown to promote differentiation and morphogenesis of many different cell types, including tubular cells. This study investigated the growth, differentiation, and tubular morphogenesis of human eccrine sweat gland epithelial cells cultured in Matrigel. Human eccrine gland epithelial cells were isolated and cultured in vitro. The cell growth in Matrigel was evidenced by the formation of cell clusters, which were observed under an inverted microscope. The internal structure of the cell clusters was further investigated by hematoxylin-eosin (HE) staining and confocal laser scanning microscopy (CLSM) of propidium iodide-stained nuclei. The results demonstrated that although on a plastic surface or in a collagen gel the cells could not form tubular structures, they formed tubular structures when cultured in Matrigel. Consequently, we conclude that Matrigel can promote tubular morphogenesis of human eccrine sweat gland epithelial cells.

405 VAV3, A POSSIBLE KEY MOLECULE, ENHANCES ANDROGEN-INDEPENDENT GROWTH AND THE MALIGNANT POTENTIAL OF PROSTATE CANCER CELLS UNDER CHRONIC HYPOXIA

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011405 VAV3, A POSSIBLE KEY MOLECULE, ENHANCES ANDROGEN-INDEPENDENT GROWTH AND THE MALIGNANT POTENTIAL OF PROSTATE CANCER CELLS UNDER CHRONIC HYPOXIA Kenichi Hirai, Takeo Nomura, Mutsushi Yamasaki, Takanori Matsubara, Fuminori Sato, Masatsugu Moriyama, and Hiromitsu Mimata Kenichi HiraiKenichi Hirai Oita, Japan More articles by this author , Takeo NomuraTakeo Nomura Oita, Japan More articles by this author , Mutsushi YamasakiMutsushi Yamasaki Oita, Japan More articles by this author , Takanori MatsubaraTakanori Matsubara Oita, Japan More articles by this author , Fuminori SatoFuminori Sato Oita, Japan More articles by this author , Masatsugu MoriyamaMasatsugu Moriyama Oita, Japan More articles by this author , and Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.494AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hypoxia occurs in many solid tumors, including prostate cancer, and enhances tumor progression and treatment resistance. Our previous studies showed that chronic hypoxia promoted androgen-independent growth and malignant cell behavior in a human prostate cancer cell line. From the results of multi-pathway analysis of gene expression of LNCaP cells under a chronic hypoxic condition, we specifically focused on the biological functions of Vav3 gene in vitro. METHODS We analyzed the gene expression profiles of LNCaP cells under normoxic, acute hypoxic (1% oxygen for forty-eight hours), and chronic hypoxic conditions (cultured over six months). Comprehensive mRNA profiling was performed using an Agilent 44K array platform, and we investigated the association between elevated genes and tumor behavior by the applications of Ingenuity Pathways Analysis. From the results, we identified the Vav3 gene and closely analyzed the details of the gene in vitro. We validated the expression by quantitative RT-PCR and Western blot analyses. To analyze the function of Vav3, knockdown by siRNA was performed. Next, we transfected LNCaP with the Vav3 cDNA expression vector (control cells were transfected with neo expression vector) and isolated stable clones with high or low expression. The growth of cancer cells was evaluated in vitro. Also, we examined cell invasiveness and migration using the Matrigel invasion assay. For cell cycle analysis, flow cytometric analysis of propidium iodide-stained nuclei was carried out. RESULTS The results were as follows: (1) Vav3 mRNA and protein expression levels were increased in LNCaP cells under chronic hypoxia, respectively; (2) chronic hypoxia increased both the invasiveness and migration of LNCaP cells; (3) LNCaP cells under chronic hypoxia showed less invasiveness and cell motility as well as suppressed their growth rates by interrupting Vav3 expression using Vav3 siRNA; (4) Vav3 expression positively correlated with the growth, invasiveness, and motility of LNCaP cells; and (5) interrupting the Vav3 signaling axis with siRNA led to the apoptosis of LNCaP cells via the mitochondrial pathway, with increased activation of caspases-3, and -9 and cleaved PARP. CONCLUSIONS Our results showed that chronic hypoxia promoted the malignant potential of prostate cancer cells via the overexpression of Vav3. Therefore, targeting the Vav3 signaling pathway could prove to be a promising approach for the treatment of progressive or castration resistant prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e163-e164 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenichi Hirai Oita, Japan More articles by this author Takeo Nomura Oita, Japan More articles by this author Mutsushi Yamasaki Oita, Japan More articles by this author Takanori Matsubara Oita, Japan More articles by this author Fuminori Sato Oita, Japan More articles by this author Masatsugu Moriyama Oita, Japan More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

406 HIGH-THROUGHPUT SCREEN FOR GENES THAT SELECTIVELY PROMOTE GROWTH OF ANDROGEN INDEPENDENT PROSTATE CANCER CELLS

CaP cells under normoxic, acute hypoxic (1% oxygen for forty-eight hours), and chronic hypoxic conditions (cultured over six months). Comprehensive mRNA profiling was performed using an Agilent 44K array platform, and we investigated the association between elevated genes and tumor behavior by the applications of Ingenuity Pathways Analysis. From the results, we identified the Vav3 gene and closely analyzed the details of the gene in vitro. We validated the expression by quantitative RT-PCR and Western blot analyses. To analyze the function of Vav3, knockdown by siRNA was performed. Next, we transfected LNCaP with the Vav3 cDNA expression vector (control cells were transfected with neo expression vector) and isolated stable clones with high or low expression. The growth of cancer cells was evaluated in vitro. Also, we examined cell invasiveness and migration using the Matrigel invasion assay. For cell cycle analysis, flow cytometric analysis of propidium iodide-stained nuclei was carried out. RESULTS: The results were as follows: (1) Vav3 mRNA and protein expression levels were increased in LNCaP cells under chronic hypoxia, respectively; (2) chronic hypoxia increased both the invasiveness and migration of LNCaP cells; (3) LNCaP cells under chronic hypoxia showed less invasiveness and cell motility as well as suppressed their growth rates by interrupting Vav3 expression using Vav3 siRNA; (4) Vav3 expression positively correlated with the growth, invasiveness, and motility of LNCaP cells; and (5) interrupting the Vav3 signaling axis with siRNA led to the apoptosis of LNCaP cells via the mitochondrial pathway, with increased activation of caspases-3, and -9 and cleaved PARP. CONCLUSIONS: Our results showed that chronic hypoxia promoted the malignant potential of prostate cancer cells via the overexpression of Vav3. Therefore, targeting the Vav3 signaling pathway could prove to be a promising approach for the treatment of progressive or castration resistant prostate cancer.

First nuclear DNA amounts in diploid (2n = 2x = 14) Corchorus spp. by flow cytometry: genome sizes in the cultivated jute species (C. capsularis L. and C. olitorius L.) are ∼300% smaller than the reported estimate of 1100–1350 Mb

We report the 2C values (nuclear DNA content) and haploid genome (1C) sizes of the Corchorus species (2n = 2x = 14), with a special reference to the two cultivated jute species, viz., C. capsularis L. (the white jute) and C. olitorius L. (the tossa jute). Flow cytometric analysis of propidium iodide-stained nuclei showed that the 2C values were between 0.502 and 0.695 pg for C. capsularis, and between 0.643 and 0.718 pg for C. olitorius. For both the cultivated species, however, mutants had higher amounts of nuclear DNA than that of the cultivars. Amongst the wild Corchorus species, the lowest 2C value was observed in C. fascicularis (0.384 pg), while the highest in C. pseudo-olitorius (0.712). The current 2C values of both the cultivated jute species are in disagreement with earlier estimates of 2.3 and 2.8 pg in C. capsularis and C. olitorius, respectively. Of the Corchorus species studied, C. fascicularis has the smallest haploid genome (~188 Mb) followed by C. aestuans (~194 Mb). Our results show that, on an average, genome sizes (1C values) of C. capsularis and C. olitorius are ~280 Mb and ~324 Mb, respectively, which are ~300% smaller than their corresponding reported estimates. The white jute cv. JRC-212 (or Branca) has the smallest genome (~246 Mb) among the economically important crop species.

A Novel Roscovitine Derivative Potently Induces G1-Phase Arrest in Platelet-Derived Growth Factor-BB-Activated Vascular Smooth Muscle Cells

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine (LGR1406), a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC. Cell proliferation was quantified measuring DNA synthesis via 5-bromo-2'-deoxyuridine incorporation. Analysis of cell cycle distribution was done by flow cytometry using propidium iodide-stained nuclei. Key regulators of the cell cycle and relevant signaling pathways were dissected by Western blot analyses. In addition, in vitro kinase assays and in silico studies regarding the pharmacokinetic profile of both compounds were performed. LGR1406 shows a stronger (IC(50) = 3.0 muM) antiproliferative activity than ROSC (IC(50) = 16.9 muM), halting VSMCs in G(0)/G(1) phase of the cell cycle, whereas ROSC does not arrest but rather delays cell cycle progression. Neither of the compounds interferes with early PDGF-BB-induced signaling pathways (p38, extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, Akt, signal transducer and activator of transcription 3), and both inhibit CDKs, with LGR1406 exerting a slightly higher potency against CDK1/2 and 4 than ROSC. Expression of cyclins A and E as well as hyperphosphorylation of the pocket proteins retinoblastoma protein and p107 are negatively affected by both compounds, although to a different extent. In silico calculations predicted a much higher metabolic stability for LGR1406 compared with ROSC. Altogether, ROSC derivatives, such as LGR1406 seem to be promising compounds for further development in antirestenotic therapy.

The nuclear genome of the phytoseiid Metaseiulus occidentalis (Acari: Phytoseiidae) is among the smallest known in arthropods

The genome size of the phytoseiid Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) needs to be estimated before the whole nuclear genome can be sequenced. Two different procedures were used to estimate the genome size of M. occidentalis; (1) flow cytometry (Marescalchi et al. in Genome 33:789-793, 1990) and (2) quantitative real-time PCR (qRT-PCR) (Wilhelm et al. in Nucleic Acids Res 31:e56, 2003). Fluorescence intensity of propidium iodide-stained nuclei of M. occidentalis was measured by flow cytometry using females, males, and eggs. Only the eggs yielded peaks, which ranged in size from 35 to 160 Mb, with a tall peak of 140 Mb in 1-day-old eggs and 65 Mb in 2-day-old eggs, respectively. However, the peaks are broad and do not provide an accurate estimate. The qRT-PCR procedure required single-copy nuclear gene sequences from this phytoseiid. This was accomplished by designing degenerate primers, amplifying the Actin and EF1alpha sequences from M. occidentalis, and then designing M. occidentalis-specific primers that amplified a unique sequence. The standard qRT-PCR protocol was inefficient and amplification failed frequently, so we developed a high-fidelity qRT-PCR protocol, which utilizes a mix of two DNA polymerases (Taq and a proof-reading Tgo or ACCUZYME) to consistently amplify sequences. This allowed us to estimate the nuclear genome size of M. occidentalis as 88-90 +/- 5 Mb. When compared to other arthropod genomes, this appears to be very small.

Vitamin D3 Induces Autophagy of Human Myeloid Leukemia Cells

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.

Nuclear DNA content variation in Halimium and Xolantha (Cistaceae)

Chromosome numbers and nuclear DNA contents were determined in several taxa of two genera of Cistaceae, Halimium and Xolantha. The diploid chromosome number 2n = 18 is stable within the genus Halimium. In Xolantha, the diploid chromosome number 2n = 14 was confirmed in X. tuberaria, and hexaploid (2n = 36) and octoploid (2n = 48) cytotypes were found in X. guttata. DNA amounts, first reported here for these genera, were determined by flow cytometry of propidium iodide-stained nuclei. Within Halimium there is a more than twofold difference in genome size between H. calycinum (7.61 pg DNA) and H. atriplicifolium (3.66 pg DNA). Within the genus Xolantha, absolute genome sizes of 7.3 and 9.0 pg DNA were determined for the hexaploid and octoploid cytotypes of X. guttata, respectively, but the mean 2C-values of this polyploid species (2.31) and the diploid X. tuberaria (3.34) are significantly lower than those of Halimium or Cistus.

Cyclin-dependent Kinase Inhibitor Roscovitine Induces Cell Cycle Arrest and Apoptosis in Rabbit Retinal Pigment Epithelial Cells

Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells. Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot. Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine. This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

Functional role of bone morphogenetic protein-4 in isolated canine parietal cells

Bone morphogenetic protein (BMP)-4 is an important regulator of cellular growth and differentiation. Expression of BMP-4 has been documented in the gastric mucosa. We reported that incubation of canine parietal cells with EGF for 72 h induced both parietal cell morphological transformation and inhibition of H(+)/K(+)-ATPase gene expression through MAPK-dependent mechanisms. We explored the role of BMP-4 in parietal cell maturation and differentiation. Moreover, we investigated if BMP-4 modulates the actions of EGF in parietal cells. H(+)/K(+)-ATPase gene expression was examined by Northern blots and quantitative RT-PCR. Acid production was assessed by measuring the uptake of [(14)C]aminopyrine. Parietal cell apoptosis was quantitated by Western blots with anti-cleaved caspase 3 antibodies and by counting the numbers of fragmented, propidium iodide-stained nuclei. MAPK activation and Smad1 phosphorylation were measured by Western blots with anti-phospho-MAPK and anti-phospho-Smad1 antibodies. Parietal cell morphology was examined by immunohistochemical staining of cells with anti-H(+)/K(+)-ATPase alpha-subunit antibodies. BMP-4 stimulated Smad1 phosphorylation and induced H(+)/K(+)-ATPase gene expression. BMP-4 attenuated EGF-mediated inhibition of H(+)/K(+)-ATPase gene expression and blocked EGF induction of both parietal cell morphological transformation and MAPK activation. Incubation of cells with BMP-4 enhanced histamine-stimulated [(14)C]aminopyrine uptake. BMP-4 had no effect on parietal cell apoptosis, whereas TGF-beta stimulated caspase-3 activation and nuclear fragmentation. In conclusion, BMP-4 promotes the induction and maintenance of a differentiated parietal cell phenotype. These findings may provide new clues for a better understanding of the mechanisms that regulate gastric epithelial cell growth and differentiation.

112 VIABILITY AND APOPTOSIS OF BOVINE FIBROBLASTS FROZEN IN A CONVENTIONAL ( - 20°C) OR LOW-TEMPERATURE ( - 80°C) FREEZER

Fibroblasts are commonly frozen in dimethyl sulfoxide (DMSO, 10% v/v) at a cooling rate of 1°C min−1 in cryo-containers placed in a low-temperature (−80°C) freezer (LTF) overnight before storage in liquid nitrogen (LN2). LTFs are not always available and cell viability may be altered by freezing at different cooling rates. Therefore, the purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen at different cooling rates in a −80°C freezer or conventional −20°C freezer. Fibroblasts were generated from skin tissue collected from an adult female cow. Tissue was cut and cultured in DMEM for 7 to 10 days and passaged 2 or 3 times before being cryopreserved. Cells were frozen by one of 6 procedures; cells that were not frozen served as a control. To be frozen, cells were resuspended in DMEM + 10% FBS and 10% DMSO in 2-mL cryo-vials before being placed into freezing containers (Mr. Frosty, Nalgene). For groups 1 and 2, the container was placed in a −20°C freezer and cooled at ≈0.6°C min−1 to −7°C; ice formation was induced by seeding, after which cooling was continued at ≈0.6°C min−1 to −20°C. Vials in groups 1 and 2 were placed in LN2 after 1 h and 16 to 18 h, respectively. Group 3 and 4 vials were also cooled to −20°C at 0.6°C min, except that these samples were not seeded. After being held at −20°C for 16 to 18 h, group 3 vials were thawed; group 4 vials were stored in LN2. Vials in groups 5 and 6 were placed into a Mr. Frosty container and cooled at ≈1°C min−1 in a −80°C freezer. After being held at −80°C for 16 to 18 h, group 5 vials were thawed, and group 6 vials were placed into LN2. Group 1, 2, 3, and 4 vials were thawed in a 37°C water bath; group 5 and 6 vials were thawed in a 40°C water bath. Cell viabilities were quantified by using TB exclusion (TB) with microscopy. At least 20 000 cells from each group were thawed, and caspase 3, a key enzyme involved in apoptotic processes, was detected using a Caspase 3-FLICA apoptosis detection kit; propidium iodide-stained nuclei were assessed with cytometry (FCM) to determine viability (Table 1). Data were analyzed by ANOVA (P &amp;lt; 0.05). Although there were viability differences among groups, freezer type, seeding, holding time, and LN2 had no significant impact, as assessed by TB or FCM; however, no LN2 significantly increased the percentage of viable cells displaying apoptosis. In summary, bovine fibroblasts were successfully frozen in a conventional (−20°C) freezer before storage in LN2. Table 1. Viability and apoptosis of bovine fibroblasts frozen at −20°C or −80°C

Differential polyamine analogue effects in four human breast cancer cell lines

Polyamine analogues have demonstrated anti-tumour activity in a number of solid tumour models. In the present study we compared the cytotoxicities of three polyamine analogues against four breast cancer cell lines. All cell lines are derived from tumours of women with breast cancer and, although we are sampling just a small number of tumours, they represent a spectrum of the genetic plethora of breast cancers. Cytotoxicity, over a dose range from 0.1 to 100 μM, was evaluated with three different cytotoxicity assays performed in 96-well plates. Comparing the effects of the analogues on polyamine pools with data from the cytotoxicity assays indicates that there was not a direct correlation between polyamine pool depletion and cytotoxicity. Flow cytometry was used to investigate analogue-induced cell death as measured by the appearance of a sub-G 1 peak. Induction of cell death by the analogues differed in the cell lines, however, cell death when induced was apoptotic, as demonstrated by detection of apoptotic bodies with immunofluorescence microscopy of propidium iodide-stained nuclei. Comparing the flow cytometry-derived data and the data from the cytotoxicity assays reveals that the analogues exert their effects by inhibiting cell growth and/or inducing cell death.

Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

IntroductionRapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G1 arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin.MethodWe evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model.ResultsWe found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G1 phase, rather than apoptosis, and induced senescence-associated β-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G2, transforming growth factor-β-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects.ConclusionRapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A.

Indole-3-carbinol induces a G1 cell cycle arrest and inhibits prostate-specific antigen production in human LNCaP prostate carcinoma cells.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, is a promising anticancer agent for certain reproductive tumor cells. The objective of the current study was to characterize the cell cycle effects of I3C in human prostate carcinoma cells. The incorporation of [(3)H]thymidine and flow cytometry of propidium iodide-stained nuclei were used to monitor I3C-regulated changes in prostate carcinoma cell proliferation and cell cycle progression. Western blotting was used to document expression changes in cell cycle components and prostate-specific antigen (PSA) levels. The enzymatic activities of cyclin-dependent kinases (CDK) were tested by in vitro protein kinase assays using the retinoblastoma protein as a substrate. I3C suppressed the growth of LNCaP prostate carcinoma cells in a dose-dependent manner by inducing a G1 block in cell cycle progression. I3C selectively inhibited the expression of CDK6 protein and transcripts and strongly stimulated the production of the p16 CDK inhibitor. In vitro protein kinase assays revealed the striking inhibition by I3C of immunoprecipitated CDK2 enzymatic activity and the relatively minor down-regulation of CDK4 enzymatic activity. In LNCaP prostate carcinoma cells, I3C treatment inhibited production of PSA, whereas combinations of I3C and the androgen antagonist flutamide more effectively inhibited DNA synthesis and PSA levels compared with either agent alone. The results of the current study demonstrated that I3C has a potent antiproliferative effect in LNCaP and other human prostate carcinoma cells. These findings implicate this dietary indole as a potential chemotherapeutic agent for controlling the growth of human prostate carcinoma cells.

The Prognostic Significance of the Overexpression of HER-2/ neu in Korean Gastric Carcinomas and the In Vitro Effects of Anti-HER-2/neu Antibody on Cell Growth in the Gastric Carcinoma Cell Lines.

The HER2 gene encodes a 185-kd transmembrane glycoprotein receptor (p185(HER2)) that has partial homology with the epidermal growth factor receptor (EGFR) and shares intrinsic tyrosine kinase activity. The HER2 gene has been found to be amplified in various human cancers and to be associated with poor prognosis. The authors investigated the correlation between clinicopathologic factors and the overexpression of the p185(HER2) in Korean gastric adenocarcinoma patients, and determined whether the antiproliferative effects of anti- p185(HER2) antibody can also be observed on gastric cancer cell lines that overexpress this growth factor receptor. We evaluated the relationship between p185(HER2) overexpression and clinicopathological features in 94 (M: F=52: 42) gastric adenocarcinoma patients (median age 59 years). Protein expression was analysed by immunohistochemical staining in paraffin embedded tissues with monoclonal antibody for p185(HER2). To explore the role of humanized anti-p185(HER2) monoclonal antibody trastuzumab (Herceptin ) in vitro, the growth curve of Korean gastric cancer cells that overexpress the p185(HER2) protein was studied and a cell cycle analysis was performed. p185(HER2) overexpression correlates positively with lymph node metastasis (p=0.002), distant metastasis (p=0.01), AJCC classification (p=0.01), higher relapse rate p=0.001), and a tendential association with the pT stage (p=0.054). p185(HER2) overexpression was found to be more frequent in advanced gastric cancer than early gastric cancer (54.1% vs 24.2%, p=0.008). Patients with overexpression of p185(HER2) were found to have significantly lower relapse-free (p=0.003) and overall survival (p= 0.0004) than patients without overexpression. Among several Korean gastric cancer cell lines, SNU-1, SNU-5, and SNU-620 overexpress p185(HER2). Trastuzumab inhibited the proliferation of p185(HER2) overexpressed Korean gastric cancer cell line by 21% with down-regulation of p185(HER2) protein expression. DNA fluorescence flow cytometry of propidium iodide-stained nuclei showed a reduction in the fraction of the S phase following treatment with trastuzumab. S: Taken together, our observations suggest the potential prognostic significance of p185(HER2) overexpression in Korean gastric adenocarcinoma patients and point to the need for further research on this mechanism. This suggests the possible use of p185(HER2) as a therapeutic target in gastric cancer.