Neural cell adhesion molecule mediates contact-dependent inhibition of growth of near-diploid mouse fibroblast cell line m5S/1M

A near-diploid mouse fibroblast cell line m5S/1M used in this study shows a high sensitivity to contact-dependent inhibition of growth, and the addition of EGF causes both morphological change and loss of contact-dependent inhibition of growth. The m5S/1M cell and its transformants obtained by x-ray irradiation have been used to search for the cell surface glycoproteins that are responsible for the growth regulation via cell-cell interactions. Lectin blotting analyses showed that the expression of the cell surface glycoprotein of 140 kD (140KGP) is highly sensitive to the transformation induced either by x-ray irradiation or by the EGF stimulation. We purified the 140KGP and found that it was composed of two glycoproteins. The major component of 140KGP was identified as neural cell adhesion molecule (NCAM) by amino acid sequence analyses of the peptide fragments and by the cross-reactivity with anti-NCAM mAb, clone H28.1.2.3. Monoclonal antibody against 140KGP (clone LN-10) recognizes all three isoforms of NCAM expressed on m5S/1M cell and showed that the expression of NCAM was highly sensitive to the transformation. Furthermore, the immobilized LN-10 strongly inhibited the growth of actively proliferating m5S/1M cells and the LN-10 in a soluble form showed a significant growth-stimulating effect on the confluent quiescent cultures of m5S/1M cells. The results show that NCAM plays a major role in the contact-dependent inhibition of growth of m5S/1M, and that NCAM might be involved in the regulation of cell growth during embryogenesis and formation of nervous systems.     

Cell transformation as aberrant differentiation：Environmentally dependent spontaneous transformation of NIH 3T3 cells

NIH 3T3 cells, a mouse fibroblast cell line used as routine target cells for transfection experiments, undergo spontaneous transformation in our experiments after they form a confluent sheet in medium containing fetal bovine serum (FBS) or lower coneentration of calf serum (CS). The transformation takes the form of foci of multiplying cells among the surrounding cells which have stopped cell division. However, no focus of transformed cells could be seen in medium containing high concentration (10％) of CS. Further experiments indicated that the frequency of transformation is highly dependent on the concentration of serum and the transformation in CS is changeable when the cells are passaged in FBS. 3~H-thymidine autoradiography has been proved to be a sensitive measurement indicator for focus formation. Our results suggest that the high frequency of transformation and its dependence on confluency as well as on medium composition are characteristics of cell differentiation rather than mutation. The role of the NIH 3T3 cell line as a cancer-initiated cell population and its accelerated transformation by ras oncogene might be considered as a form of tumor promotion is discussed.     

A mammalian somatic "cell cycle" mutant defective in G1

Variants or “mutants” temperature-sensitive (ts) for growth have been isolated by selection from a near-diploid mouse cell line. Thus far. 10 ts mutants which grow normally at 33° C, but not at 39° C, have been isolated. These ts mutants were then studied to determine if any manifested their defect at a unique point or stage in the cell cycle. This type of ts mutant is termed a “cell cycle” mutant. The first screen involves observing individual cells of an asynchronous culture for residual division after a shift from 33° C (permissive temperature) to 39° (nonpermissive temperature). A cell cycle mutant should show some fraction of the cells dividing only once at a normal rate after the shift. The ts variant B54 met this first criterion for a cell cycle mutant (i.e., 50% residual division) and was further analyzed. The second screening technique monitors (1) the rate of entry into S, (2) the length of G₂, and (3) the rate and duration of cells entering mitosis after a shift of an asynchronous culture to 39°. This experiment with B54 revealed that cells in G₁ at the time of the shift to 39° failed to enter S while cells already into S completed the cycle at 39°. These results suggest that B54 is defective in a G₁ function which is required for entry into S, but which is no longer needed once cells have entered S. Other results are presented which also support this hypothesis. In addition the ts function of B54 is apparently required for recovery from a “high density” G₁ arrest.     

Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine

We have previously shown that 5-azacytidine (5-Aza-CR) induced the formation of biochemically differentiated myotubes, adipocytes, and chondrocytes in the mouse embryo cell line, C3H/10T1/2CL8 (10T1/2), and that the induction of the muscle phenotype was cell cycle specific. Here we show that the adipocyte phenotype is also induced maximally in cells treated during early S phase. During this period, the minimum treatment time required for the subsequent formation of myotubes was 5 min and the number of myotubes formed was dependent on treatment time. The incorporation of ¹⁴C-5-Aza-CR into DNA during the cell cycle, however, was not enhanced during early S phase, suggesting that incorporation of 5-Aza-CR into specific DNA sequences synthesized during early S phase may be required for the expression of the new phenotypes. Single cells, obtained by plating cell suspensions into 16 mm wells at limiting dilution, were treated with 5-Aza-CR during S phase. The resulting clones showed a high frequency of phenotypic conversion, indicating that 5-Aza-CR did not act via a selective mechanism, and several of the clones were capable of expressing more than one phenotype. The cells required more than 2 division cycles after treatment with the analog for the expression of the muscle phenotype and the capacity to differentiate was retained for long periods of time in the absence of cell division. The adult mouse line, CVP3SC6, differentiated into functional striated muscle cells following treatment with 5-Aza-CR. The analog also caused oncogenic transformation in the adult line at the same concentration that was effective at inducing myogenic expression.     

Isolation and characterization of a near-diploid differentiated cell line from a murine teratocarcinoma that differentiates into muscle

Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e.g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice.     

Isolation and characterization of a near-diploid differentiated cell line from a murine teratocarcinoma that differentiates into muscle

Summary Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e. g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice. This work was supported in part by National Institutes of Health, Bethesda, MD, Grant CA-15823 and by a departmental gift from R. J. Reynolds Industries, Inc.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.     

Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Transformation of normal diploid cells by isolated metaphase chromosomes of virus-transformed or spontaneous tumor cells.

Normal diploid human cells with a limited life-span in culture, as well as primary or secondary cell cultures of mouse or rat embryos, can be transformed in vitro (i.e. grow in soft-agar or low-serum medium) after a single exposure to metaphase chromosomes from SV40-transformed human or rat cells, Ad5-transformed human cells and several spontaneous human or mouse tumor cells. Chromosomes from normal diploid cells do not show any such transforming activity. As judged from the number of colonies formed in selective medium, the efficiency of transformation is, with some exceptions, of the order of 10(-5)--10(-6) and is generally higher for homologous than for heterologous transfers. A fraction of the colonies demonstrate abortive transformation. Nevertheless, using chromosomes from all but one donor cell population, at least one transferent cell line expressing a stable transformed phenotype has been established. Our results demonstrate that transformation of normal diploid cells by a presumptive chromosome-mediated gene transfer can be obtained with a variety of donor and recipient cells.     

Characterization of a temperature-sensitive mutant of BALB-c 3T3 cells

A temperature-sensitive mutant, designated ts-1, has been isolated from BALB/c 3T3 mouse cells; this is the first such mutant of the cell line to be reported. The mutant is similar to the original cell line in morphology, growth rate, and contact inhibition at the permissive temperature (33 °C). When ts-1 is studied at the non-permissive temperature (38-38.5 °C), however, cell division ceases. Incorporation of radioactive thymidine or uridine under conditions in which the amount of isotope incorporated reflects the amount of the appropriate macromolecule present in the culture shows gradual cessation corresponding to the inhibition of cell division. On the other hand, in pulse experiments, incorporation of radioactive thymidine or uridine continues, although at a diminished rate (50%). These results suggest substantial turnover of DNA and RNA at the non-permissive temperature.The cessation of growth of ts-1 upon shift to 38.5 °C is markedly dependent on the cell number at the time of shift. Experiments in which cell number is kept constant but initial area of inoculation or volume of medium are varied indicate that the cell number per unit area is most important. Though the biochemical basis for this ‘cell-cooperation’ is unknown, these results might explain the apparent low incidence of mutants recovered in our studies thus far.     

Induction of ouabain-resistance mutation and cycle-dependent transformation of C3H/10T1/2 cells by N-nitroso-2-acetylaminofluorene

Ouabain-resistance mutation and cell cycle-dependent transformation were studied concurrently in the C3H/10T1/2 cell line treated with N-nitroso-2-acetylaminofluorene (N-NO-AAF) or N-nitroso-N-2-fluorenylacetamide. N-NO-AAF is a new direct-acting mutagen that exhibits a very short half-life (34 min) in complete medium independent of cell number seeded. With 0.1-0.3 mM of N-NO-AAF, cytotoxicity was noted after exposure for 2 h, but another phase of cytotoxicity was observed between 8 and 24 h. N-NO-AAF was more toxic than its parent compound 2-AAF. Moreover, maximal mutation frequency at the Na+/K(+)-ATPase gene locus (ouar mutation) was attained within 30 or 40 min of exposure, dependent on dosage of N-NO-AAF. With 2-AAF, 2-AF and 2-nitrofluorene, however, no detectable mutants were found under the same conditions. In cell cycle-dependent transformation assays, cells were synchronized by release from confluence-induced arrest of proliferation, 2 concentrations of N-NO-AAF were added for 2 h at various intervals during the cell cycle. The results clearly revealed that cells in 2 specific time intervals were susceptible to malignant transformation, i.e., at 10 and 18 h (early S phase) after release from the block.     

Diacylglycerol kinase inhibition prevents IL-2-induced G1 to S transition through a phosphatidylinositol-3 kinase-independent mechanism.

Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the alpha isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus

We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus . We have also characterized the effect of conditional Topo IV mutations on cell division and morphology. Topo IV mutants of C. crescentus are unlike mutants of Escherichia coli and S. typhimurium , which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells. Topo IV mutants of C. crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA. These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium. In addition, distinctive nucleoid-partitioning defects are not apparent in C. crescentus Topo IV mutants as they are in E. coli and S. typhimurium . However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C. crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth. We discuss these results in connection with the possible roles of C. crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis. Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family.     

Phosphorylation of histone H3 serine 10 in early mouse embryos: Active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.