FM3A Cells Research Articles

Extracellular Leakage Protein Patterns in Two Types of Cancer Cell Death: Necrosis and Apoptosis.

Dead cells release fragments of DNA, RNA, and proteins (including peptides) into the extracellular space. Two major forms of cell death during cancer development have been identified: necrosis and apoptosis. Our group investigated the mechanisms that regulate cell death during the treatment of mouse tumor FM3A cells with the anticancer drug floxuridine (FUdR). In the original strain F28-7, FUdR induced necrosis, whereas in the variant F28-7-A, it induced apoptosis. Here, we report that the extracellular leakage proteome (i.e., the secretome) is involved in these cell death phenomena. The secretome profile, which was analyzed via shotgun proteomic analysis, revealed that altered protein leakage was involved in signal transduction, transcription, RNA processing, translation, and cell death. Notably, the characteristic secretory proteins high mobility group box 1 and 2 were detected in the culture medium of both necrotic and apoptotic cells. Overall, these results indicate that unique cellular events mediated by secretory proteins may be involved in necrosis and apoptosis.

Seeking an Important Role on Metabolomics-Effects of β-Estradiol on Lipoprotein Metabolism in Mammary Tumors

The role of β-estradiol (E2) in lipoprotein metabolism in mammary tumors remains unknown. Therefore the effect of E2 on secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells was examined. The E2-treated FM3A cells increased active LPL secretion in a time- and dose-dependent manner. The activity of mitogen-activated protein kinase (MAPK) was elevated in the tumor cells treated with E2, and E2-stimulated secretion of LPL was suppressed by the MAPK kinase 1/2 inhibitor PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor FR180204, p38 MAPK inhibitor SB202190, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In addition, the effect of E2 on active LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but not by the mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed the secretion of LPL by E2. Stimulatory secretion of LPL by E2 from the tumor cells is closely associated with activation of mTORC2 rather than mTORC1, possibly via the MAPK cascade.

Palbociclib regulates intracellular lipids in mammary tumor cells by secreting lipoprotein lipase.

Lipoprotein metabolism is essential for the growth and proliferation of cancer cells, and is involved in the supply of energy and cellular components. Lipoprotein lipase (LPL) is a very important enzyme in lipoprotein metabolism; however, the details underlying the mechanism of LPL secretion are unclear. Palbociclib is an antitumor drug that inhibits cell cycle progression and suppresses the growth of cancer cells. The effects of palbociclib on energy metabolism, particularly on lipid metabolism, have not been fully elucidated. We examined the regulation of LPL secretion, which is primarily involved in lipoprotein metabolism. FM3A mouse mammary tumor cells, which are hormone receptor-positive breast cancer cells, were treated with palbociclib, and the activity and protein levels of secreted LPL were measured. Moreover, the changes in intracellular lipid content were measured by fluorescence staining using Nile Red. FM3A cells were treated with palbociclib, the activity and protein content of secreted LPL were increased. The stimulatory secretion of LPL by palbociclib was suppressed by an intracellular Ca2+ chelator (BAPTA-AM) and a Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor (STO-609). Furthermore, the palbociclib-stimulated secretion of LPL was not observed in AMP-activated protein kinase (AMPK)-knockdown cells. An increase in the fluorescence intensity of Nile Red was observed in palbociclib-treated cells; however, no increase was observed in LPL-knockdown cells. Our data suggest that palbociclib causes intracellular lipid accumulation in breast cancer cells by stimulating Ca2+/CaMKK/AMPK-mediated LPL secretion.

Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells.

Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.

Structural change and degradation of cytoskeleton due to the acrolein conjugation with vimentin and actin during brain infarction.

We have found recently that dendritic spine extension is inhibited through acrolein conjugation with α- and β-tubulin proteins during brain infarction. In this current study, we looked for other acrolein-conjugated proteins in the 100,000g precipitate fraction, to clarify how cytoskeleton structure is modified by acrolein. Acrolein-conjugated proteins were sought from acrolein-treated mouse FM3A and Neuro2a cells and from tissues isolated from mouse brain infarction. It was found that vimentin was conjugated with acrolein, and the conjugated amino acid residue was Cys328, which is the only Cys residue in vimentin. It was also found that Cys207, 257, 285, and Lys118 in actin, another cytoskeleton protein, were conjugated with acrolein. The structure and localization of vimentin and actin filaments were changed greatly in infarct brain in photochemically induced thrombosis model mice and in acrolein-treated Neuro2a cells. In addition, degradation of cytoskeleton proteins was accelerated in the order vimentin > tubulin > actin in mouse brain infarction. These findings indicate that a dysfunction of the cytoskeleton by acrolein is strongly involved in the tissue damage during brain infarction, together with the apoptosis caused by glyceraldehyde-3-phosphate dehydrogenase and protein degradation by matrix metalloproteinase-9.

β-Estradiol Enhanced Secretion of Lipoprotein Lipase from Mouse Mammary Tumor FM3A Cells.

The role of β-estradiol (E2) in lipoprotein metabolism in mammary tumors is unclear, therefore, we investigated the effect of E2 on the secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells. E2-treated cells increased the secretion of active LPL from FM3A cells in a time- and dose-dependent manner. Activity of mitogen-activated protein kinase (MAPK) was increased in the tumor cells treated with E2, and enhanced secretion of LPL was suppressed by MAPK kinase 1/2 inhibitor, PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor, FR180204, p38 MAPK inhibitor, SB202190, and phosphatidyl inositol 3-kinase (PI3K) inhibitor, LY294002. In addition, the effect of E2 on LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but were not by a mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed secretion of LPL by E2. These results suggest that the stimulatory secretion of LPL by E2 from the tumor cells is closely associated with an activation of mTORC2 rather than mTORC1 possibly via the MAPK cascade.

Anticancer Strategy Targeting Cell Death Regulators: Switching the Mechanism of Anticancer Floxuridine-Induced Cell Death from Necrosis to Apoptosis.

Cell death can be broadly characterized as either necrosis or apoptosis, depending on the morphological and biochemical features of the cell itself. We have previously reported that the treatment of mouse mammary carcinoma FM3A cells with the anticancer drug floxuridine (FUdR) induces necrosis in the original clone F28-7 but apoptosis in the variant F28-7-A. We have identified regulators, including heat shock protein 90, lamin-B1, cytokeratin-19, and activating transcription factor 3, of cell death mechanisms by using comprehensive gene and protein expression analyses and a phenotype-screening approach. We also observed that the individual inhibition or knockdown of the identified regulators in F28-7 results in a shift from necrotic to apoptotic morphology. Furthermore, we investigated microRNA (miRNA, miR) expression profiles in sister cell strains F28-7 and F28-7-A using miRNA microarray analyses. We found that several unique miRNAs, miR-351-5p and miR-743a-3p, were expressed at higher levels in F28-7-A than in F28-7. Higher expression of these miRNAs in F28-7 induced by transfecting miR mimics resulted in a switch in the mode of cell death from necrosis to apoptosis. Our findings suggest that the identified cell death regulators may play key roles in the decision of cell death mechanism: necrosis or apoptosis.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

The resveratrol analogue, HS‑1793, enhances the effects of radiation therapy through the induction of anti‑tumor immunity in mammary tumor growth.

Radiotherapy can induce the infiltration of immune suppressive cells which are involved in promoting tumor progression and recurrence. A number of natural products with immunomodulating abilities have been gaining attention as complementary cancer treatments. This attention is partly due to therapeutic strategies which have proven to be ineffective as a result of tumor-induced immunosuppressive cells found in the tumor microenvironment. The present study investigated whether HS-1793, a resveratrol analogue, can enhance the anti-tumor effects by inhibiting lymphocyte damage and immune suppression by regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), during radiation therapy. FM3A cells were used to determine the role of HS-1793 in the radiation-induced tumor immunity of murine breast cancer. HS-1793 treatment with radiation significantly increased lymphocyte proliferation with concanavalin A (Con A) stimulation and reduced the DNA damage of lymphocytes in irradiated tumor-bearing mice. The administration of HS-1793 also decreased the number of Tregs, and reduced interleukin (IL)-10 and transforming growth factor (TGF)-β secretion in irradiated tumor-bearing mice. In addition, HS-1793 treatment inhibited CD206+ TAM infiltration in tumor tissue when compared to the controls or irradiation alone. Mechanistically, HS-1793 suppressed tumor growth via the activation of effector T cells in irradiated mice. On the whole, the findings of the present study reveal that HS-1793 treatment improves the outcome of radiation therapy by enhancing antitumor immunity. Indeed, HS-1793 appears to be a good therapeutic candidate for use in combination with radiotherapy in breast cancer.

Reduced Toxicity of Trichothecenes, Isotrichodermol, and Deoxynivalenol, by Transgenic Expression of the Tri101 3-O-Acetyltransferase Gene in Cultured Mammalian FM3A Cells.

In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 μM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 μM, respectively, which were higher than the values of 0.23 and 3.0 μM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.

The resveratrol analog HS-1793 enhances radiosensitivity of mouse-derived breast cancer cells under hypoxic conditions.

Tumor hypoxia is associated with treatment resistance, cell proliferation, and metastatic potential, all of which contribute to a poor prognosis. Resveratrol [RES (trans-3,4',5-trihydroxystilbene)], a naturally occurring polyphenol, is enriched in grapes and red wine. This study investigated whether the resveratrol analog HS-1793 modulates the hypoxic status and the level of perfusion in mouse breast cancer FM3A cells. Our data show that HS-1793 decreased the levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor protein under hypoxic conditions in FM3A cells. HS-1793 improved perfusion and hypoxic status in tumor tissues and inhibited angiogenesis through HIF-1α suppression in mice. Moreover, HS-1793 inhibited hypoxia-induced cancer stem cell properties and enhanced ionizing radiation-induced apoptosis in hypoxic FM3A cells. Collectively, the resveratrol analog HS-1793 might act as a potent radiosensitizer and be a useful adjuvant agent against radiotherapy-resistant hypoxic cells in solid tumors.

Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206+ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206+ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.

Toxic acrolein production due to Ca2+ influx by the NMDA receptor during stroke

Background and purposeN-Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca2+, contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. MethodsA unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. ResultsWhen the size of brain infarction was decreased by N1, N4, N8-tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca2+, the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca2+ increase. ConclusionThe results indicate that toxicity of Ca2+ during brain infarction is correlated with the increase of acrolein.

Polyphenol extract from Phellinus igniarius protects against acrolein toxicity in vitro and provides protection in a mouse stroke model.

The basidiomycetous mushroom Phellinus igniarius (L.) Quel. has been used as traditional medicine in various Asian countries for many years. Although many reports exist on its anti-oxidative and anti-inflammatory activities and therapeutic effects against various diseases, our current knowledge of its effect on stroke is very limited. Stroke is a neurodegenerative disorder in which oxidative stress is a key hallmark. Following the 2005 discovery by Igarashi’s group that acrolein produced from polyamines in vivo is a major cause of cell damage by oxidative stress, we now describe the effects of anti-oxidative extracts from P. igniarius on symptoms of experimentally induced stroke in mice. The toxicity of acrolein was compared with that of hydrogen peroxide in a mouse mammary carcinoma cell line (FM3A). We found that the complete inhibition of FM3A cell growth by 5 μM acrolein could be prevented by crude ethanol extract of P. igniarius at 0.5 μg/ml. Seven polyphenol compounds named 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl-3-buten-2one, inonoblin C, phelligridin D, inoscavin C, phelligridin C and interfungin B were identified from this ethanolic extract by LCMS and 1H NMR. Polyphenol-containing extracts of P. igniarius were then used to prevent acrolein toxicity in a mouse neuroblastoma (Neuro-2a) cell line. The results suggested that Neuro-2a cells were protected from acrolein toxicity at 2 and 5 μM by this polyphenol extract at 0.5 and 2 μg/ml, respectively. Furthermore, in mice with experimentally induced stroke, intraperitoneal treatment with P. igniarius polyphenol extract at 20 μg/kg caused a reduction of the infarction volume by 62.2% compared to untreated mice. These observations suggest that the polyphenol extract of P. igniarius could serve to prevent ischemic stroke.

Niemann-Pick disease type C2 protein induces autophagy and inhibits growth in FM3A breast cancer cells.

Some peptides that are highly conserved between insects and mammals have anti-tumor action. Screening for inhibitors of cell growth from animal fluids may provide useful clues to anti-tumor drugs. Inducers of autophagy also have anti-tumor activity. The current authors recently studied a protein found in silkworm hemolymph, Niemann-Pick disease type C2 (NPC2). This protein, which is highly conserved among eukaryotes, was found to have anti-proliferative action on a silkworm cell line. The current study found that the silkworm NPC2 protein also inhibits the growth of FM3A murine breast cancer cells. In FM3A cells, silkworm NPC2 increased phosphorylation of AMP-activated protein kinase and decreased phosphorylation of Akt and mammalian target of rapamycin, which are regulators of autophagy. This study also found that NPC2 increased the amount of microtubule-associated protein light chain 3 (LC3)-II, an autophagosome marker, in FM3A cells. Silkworm NPC2 also induced an increase in the number of LC3-dots, a marker of pre-autophagic endosomes, in FM3A cells. When silkworm NPC2 was used to inhibit FM3A cell growth, that inhibition was attenuated by chloroquine, which inhibits autophagic activity by preventing lysosomal acidification. Murine NPC2 also inhibited growth and induced autophagy in FM3A cells. These findings suggest that NPC2 is involved in the induction and/or maintenance of autophagy and may help to elucidate the mechanisms underlying other neurodegenerative disorders such as Niemann-Pick disease.

Polyamine stimulation of eEF1A synthesis based on the unusual position of a complementary sequence to 18S rRNA in eEF1A mRNA.

It is thought that Shine-Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3'-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3'-end of 18S rRNA, i.e., a CR sequence, are present at -17 to -32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70% of intact mRNAs. In eEF1A mRNA, the CR sequence was located at -33 to -39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to -22 to -28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.

Cloning and transcriptional expression of mouse mannosyltransferase IV/V cDNA, which is involved in the synthesis of lipid-linked oligosaccharides.

We cloned the mouse mannosyltransferase IV/V gene (mALG11) from FM3A cells by a bioinformatic approach. The ORF contained 1476 bp encoding 492 amino acids. The cloned mALG11 complemented the growth defect of the Saccharomyces cerevisiae ALG11Δ mutant. In addition, we detected a variant cDNA by alternate splicing that had an additional four-nucleotide ATGC insertion at base 276 of the ORF. Consequently the variant cDNA encoded a truncated protein with 92 amino acids, lacking the glycosyltransferase group-1 domain. The variant cDNA occurs in many mouse strains according to EST database searches. Moreover, we detected it in FM3A cDNA, but we did not detect any such variants in the human EST database or in HeLa cDNA, although human ALG11 (hALG11) genomic DNA has the same sequence around the intron-exon boundaries as those of mALG11 genomic DNA. Hence, we concluded that there is different transcriptional control mechanism between mALG11 and hALG11.

Effects of Femara and Tamoxifen on Proliferation of FM3A Cells in Culture

In this study, antiproliferative effects of the selective estrogen receptor modulator Tamoxifen and the aromatase inhibitor letrozole (Femara) were evaluated and compared using the FM3A cell line, originating from a C3H mouse mammary carcinoma and positive in terms of estrogen receptor (ER) expression. Cell kinetic parameters including labelling index, mitotic index and labelling index were assessed after exposure of the. FM3A cell line to 0.001μg/ml of Tamoxifen and 0.25μg/ml of Femara for 4, 8, 16 and 32 h for all parameters. The results showed that cell growth was inhibited by both agents. There was a significant decrease in labelling index and mitotic index and significant increase in apoptotic index for all experimental groups. The differences between control and all experimental groups were statistically significant (p<0.001) for all applications.

Synthesis and Evaluation of Pyrrole Polyamide- 2′-Deoxyguanosine 5′-Phosphate Hybrid

Pyrrole polyamide-2′-deoxyguanosine 5′-phosphate hybrid (Hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was found to exhibit dose-dependent inhibition of cell growth.