Mouse FM3A Cells Research Articles

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.

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.

Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Increased catalase activity in mouse cell mutants resistant to paraquat.

Paraquat is widely used as a generator of superoxide radicals in a cell. First it was shown to immediately induce premature senescence in normal human fibroblasts. To assess its defense mechanisms, we characterized three paraquat-resistant mutants from mouse FM3A cells, MPQR40-1 and MPQR40-3 isolated by a single-step selection after mutagenesis, and SPQR100-6 isolated spontaneously. All exhibited six to eight times more resistance to paraquat than the parental line. In cell-cell hybrids, their phenotypes were recessive, co-dominant, or dominant, respectively. Biochemical characterization revealed that activity and mRNA level for catalase were increased in all of the mutants. In MPQR40-3 that showed the highest degree of paraquat resistance, total SOD activity and mRNA level for Cu/Zn-SOD were also increased. These results suggest that catalase plays a major role in paraquat resistance in mouse FM3A cells.

Amplification of nuclear aldolase A in mouse cell mutants resistant to Hoechst 33342

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. AT-binding ligands such as Hoechst 33258 synergistically potentiate the effect of BrdU. We isolated mouse FM3A cell mutants resistant to Hoechst 33342 and characterized two highly resistant mutants. Two-dimensional gel electrophoresis followed by peptides sequence tags revealed that nuclear aldolase A was markedly increased in both mutants. Western blot analysis confirmed that nuclear aldolase A was increased leaving cytosolic aldolase A unaltered. Its mRNA levels were also increased in the mutants. Enforced expression of aldolase A conferred resistance to Hoechst 33342 on wild-type cells. Taken together, nuclear aldolase A was shown to somehow protect the cytotoxic effect of Hoechst 33342.

Screening of Hsp105α-binding proteins using yeast and bacterial two-hybrid systems

Hsp105α is a 105-kDa stress protein, which is expressed constitutively at especially high levels in the brain compared with other tissues in mammals, and is also induced by a variety of stressors. Recently, we have shown that Hsp105α binds to α-tubulin and prevents the heat-induced disaggregation of microtubules. To further elucidate the function of Hsp105α, we searched for Hsp105α-binding proteins by screening a mouse FM3A cell library and human and mouse brain cDNA libraries using the yeast and bacterial two-hybrid systems. We showed here that Hsp105α interacted with several cellular proteins, such as cofilin, dynein light chain 2A, α-adducin, ubiquitin activating enzyme E1, phosphoglycerate kinase 1, and platelet-activating factor acethylhydrolase α1-subunit. The interaction was validated by the results of a pull-down assay and indirect immunofluorescence analysis. The significance of Hsp105α and Hsp105α-binding proteins in cells was discussed.

Identification of α-tubulin as an hsp105α-binding protein by the yeast two-hybrid system

Hsp105α is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105α prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105α, we searched for Hsp105α-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105α using the yeast two-hybrid system and obtained α-tubulin as an Hsp105α-binding protein. Hsp105α bound directly to α-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-α-tubulin antibodies indicated that Hsp105α was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105α-overexpressing COS-7 cells. These findings suggested that Hsp105α associates with α-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.

Establishment and characterization of a mouse FM3A cell mutant resistant to topoisomerase II-inhibitor NC-190.

We established an NC-190-resistant cell line, FM/NC-R, from the murine mammary carcinoma cell line FM3A and examined some of its characteristics. FM/NC-R cells were prepared by mutagen treatment followed by exposure to NC-190 in the culture medium. FM/NC-R cells were 76.5 times more resistant against NC-190 than FM3A cells as measured by their growth in vitro. FM/NC-R cells also showed cross-resistance to etoposide with NC-190. Neither NC-190 nor etoposide increased the lifespan of FM/NC-R-bearing mice at doses that prolonged the lifespan of FM3A-bearing mice more than four times. This resistance was not due to the change in the concentration of NC-190 in the cells, and there was no change in the expression of P-glycoprotein, a drug efflux pump in the cells. NC-190 and etoposide are inhibitors of DNA topoisomerase II, but there was no difference in cellular content of DNA topoisomerase II between the two cell lines as determined by Western blot analysis. The stabilization of DNA-DNA topoisomerase II cleavable complexes induced by NC-190 was lost in FM/NC-R cells. It was found that Gly881, which is located in the ATP binding site, was replaced by Arg in topoisomerase IIalpha of FM/NC-R cells. These results indicate that the NC-190-resistant cell line FM/NC-R contains a mutated DNA topoisomerase IIalpha.

Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.

Identification of a Mutated DNA Ligase IV Gene in the X-ray-hypersensitive Mutant SX10 of Mouse FM3A Cells

The mouse carcinoma cell line SX10 is a hypersensitive mutant to x-rays and bleomycin. An earlier complementation test suggests that SX10 would belong to x-ray-cross complementing group (XRCC) 4. However, in this study, a human XRCC4 expression vector failed to complement the SX10 phenotype. Consistent with the previous report, SX10 showed the same level of DNA-dependent protein kinase activity as the wild-type SR-1. We isolated and analyzed hybrids between SX10 and human diploid fibroblast cells and found that human chromosome 13 conferred the x-ray resistance to the hybrids, suggesting that a candidate gene would be located on this chromosome. Polymerase chain reaction analysis with these hybrids and x-ray-resistant transformants obtained by introducing human chromosomes into SX10 indicated that the mutant was likely to be defective in DNA ligase IV. Sequence analysis of the DNA ligase IV gene confirmed that a defect in SX10 was attributed to a transition of G to A at nucleotide position 1413 of the gene, leading to an amino acid substitution from Trp at residue 471 to a stop codon. Revertant clones (Rev1-3) derived from SX10 showed a restored x-ray resistance; Rev1 reverted to the original nucleotide G at position 1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA ligase IV cDNA vector into SX10 restored the resistance to both x-rays and bleomycin. SX10 showed a reduced frequency of chromosomal integration of transfected DNA, but the revertants restored the frequency found in the wild-type cells. These results suggest a possible involvement of DNA ligase IV in the integration event of foreign DNA as well as a crucial role in DNA double-strand break repair.

Involvement of SAPK/JNK pathway in X-ray-induced rapid cell death of human T-cell leukemia cell line MOLT-4

We found that SAPK/JNK was phosphorylated during X-ray-induced rapid cell death of MOLT-4 cells and that acid Sphingomyelinase inhibitor D609 suppressed the rapid cell death as well as phosphorylation of SAPK/JNK. Also C2-ceramide caused phosphorylation of SAPK/JNK, followed by rapid cell death. Further we isolated X-ray-resistant radiation-hybrid clones from MOLT-4 and 50 Gy irradiated mouse FM3A cells by repeated selections with 3 Gy irradiation. One of them named Rh-1a was found resistant to X-ray- as well as C2-ceramide-induced rapid cell death. Rh-1a cells had mouse DNA but no increase in either mouse or human Bcl-2 determined by Western blotting. Accumulation of p53 after X-irradiation was similarly observed in both parental MOLT-4 and Rh-1a cells. However, contrasting to prolonged and prominent phosphorylated status of SAPK/JNK in MOLT-4 cells, Rh-1a cells exhibited short transient increase and FM3A cells showed no increase of phosphorylated status SAPK/JNK after X-irradiation. Therefore, SAPK/JNK activation is considered important in X-ray-induced rapid cell death or apoptosis of MOLT-4 cells.

Histone H2B Phosphorylation in Mammalian Apoptotic Cells: AN ASSOCIATION WITH DNA FRAGMENTATION

Histone phosphorylation was investigated in several mammalian cells undergoing apoptosis (human HL-60 and HeLa, mouse FM3A and N18 cells, and rat thymocytes). Among the four nucleosomal core histones (H2A, H2B, H3, and H4), H2B, which is not usually phosphorylated in quiescent or growing cells, was found to be phosphorylated after treatment with various apoptotic inducers. The H2B was phosphorylated around the time when nucleosomal DNA fragmentation was initiated and, like this fragmentation, was completely blocked with Z-Asp-CH(2)-DCB, an inhibitor of ICE or ICE-like caspase. The involved single phosphopeptide of H2B proved to be phosphorylatable in vitro with a protein kinase C, and the site Ser-32 was tentatively identified. Despite typical apoptotic chromatin condensation, the H3 phosphorylation was at a low level, and the sites where phosphorylation did occur did not include any mitosis-specific phosphopeptides. Phosphorylation of H4 was increased, but the other two histone proteins (H1 and H2A) were not appreciably changed. These observations imply that 1) H2B phosphorylation occurs universally in apoptotic cells and is associated with apoptosis-specific nucleosomal DNA fragmentation, 2) chromatin condensation in apoptosis occurs by a different biochemical mechanism from those operating during mitosis or premature chromosome condensation, and 3) this unique phosphorylation of H2B is a useful biochemical hallmark of apoptotic cells.

Isolation and identification of the third subunit of mammalian DNA polymerase delta by PCNA-affinity chromatography of mouse FM3A cell extracts.

Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

Association of HSP105 with HSC70 in High Molecular Mass Complexes in Mouse FM3A Cells

The 105-kDa stress proteins HSP105α and HSP105β belong to a high molecular mass heat shock protein family which has been found in organisms from yeast to mammals. Here we demonstrated the interaction of HSP105 with HSP70 family proteins in mouse FM3A cells. The association of HSP105 with HSC70 was shown by immunoprecipitation using anti-HSP105 antibody. Furthermore, when cell extracts or partially purified HSP105 fractions from nonstressed or heat-shocked cells were analyzed by size exclusion chromatography, density gradient centrifugation or cross-linking, HSP105 was detected as HSP105/HSC70 complexes with molecular masses of approximately 300-500-kDa, 160-kDa or 200-kDa, respectively. Since the 160-200-kDa complexes must be HSP105/HSC70 heterodimers, the 300-500-kDa complexes seemed to consist of HSP105/HSC70 heterotetramers possibly with other proteins. Our finding that HSP105 is complexed with HSC70 suggests that HSP105 may function cooperatively with HSC70, that HSP105 regulates the function of HSC70 or that HSC70 reversibly regulates the function of HSP105 in cells under both nonstressed and stressed conditions.

Structure and activity of mouse S-adenosylmethionine decarboxylase gene promoters and properties of the encoded proteins.

The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library. One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2). There was no sequence identity between the two promoters. The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters. After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter. Similar results were obtained by using stably transfected mouse FM3A cells. In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold. AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells. When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities. The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57. Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small. Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene.

A Mutant Strain of Mouse FM3A Cells Defective in Apoptotic DNA Fragmentation

A mutant strain of mouse FM3A cells was defective in apoptotic DNA fragmentation. Upon DNA damage by UV-radiation, the strain did not form DNA fragments, though it accumulated a large amount of p53 protein as did wild-type cells. No DNA fragmentation was observed when the strain was subjected to other kinds of DNA damaging agents such as X ray-radiation, or addition of etoposide to the culture medium. Other well-known biological stimulations inducing apoptosis, serum depletion or addition of TNF-α to the culture medium, also failed to cause DNA fragmentation in the cells. Using this apoptosis-deficient strain, we characterized another apoptotic reaction, export of phosphatidylserine to the cell surface. This molecule was exported onto the surface in both wild-type and the mutant cells when cells were treated with TNF-α and cycloheximide. Thus, the export of phosphatidylserine is suggested to be independent of the pathway for apoptotic DNA fragmentation.

Apoptosis Was Promoted at a Nonpermissive Temperature in DNA Replication-Defective Temperature-Sensitive Mutants of Mouse FM3A Cells

Apoptosis was promoted at the nonpermissive temperature in some temperature-sensitive (ts) mutant strains of mouse FM3A cells deficient in initiation of DNA replication. We examined expression of cell cycle regulation genes in the four ts mutant strains and found that two strains, tsFT107 and tsFT111, exhibited marked accumulation of p53 protein by a posttranscriptional mechanism at 16 h after temperature up-shift. These two strains also exhibited high levels of p21 mRNA expression, repression of cyclin A and D1 mRNAs, and obvious accumulation of underphosphorylated retinoblastoma protein. Only these two strains died by apoptosis at day 3 after up-shift, although no change was observed in the level of bax mRNA. These results suggest the existence of two types of responses after temperature up-shift in the four temperature-sensitive cell strains of the initiation of DNA replication: one type directs inappropriate DNA replication that then may produce endogenous DNA damage, p53-mediated cell cycle arrest, and subsequent apoptosis, while the other type exhibits only the p53-independent cell cycle arrest.

A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells.

In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.