Cultured Mouse Embryonic Fibroblasts Research Articles

Constitutive telomerase expression promotes mammary carcinomas in aging mice.

Telomerase is up-regulated in the vast majority of human cancers and serves to halt the progressive telomere shortening that ultimately blocks would-be cancer cells from achieving a full malignant phenotype. In contrast to humans, the laboratory mouse possesses long telomeres and, even in early generation telomerase-deficient mice, the level of telomere reserve is sufficient to avert telomere-based checkpoint responses and to permit full malignant progression. These features in the mouse provide an opportunity to determine whether enforced high-level telomerase activity can serve functions that extend beyond its ability to sustain telomere length and function. Here, we report the generation and characterization of transgenic mice that express the catalytic subunit of telomerase (mTERT) at high levels in a broad variety of tissues. Expression of mTERT conferred increased telomerase enzymatic activity in several tissues, including mammary gland, splenocytes, and cultured mouse embryonic fibroblasts. In mouse embryonic fibroblasts, mTERT overexpression extended telomere lengths but did not prevent culture-induced replicative arrest, thus reinforcing the view that this phenomenon is not related to occult telomere shortening. Robust telomerase activity, however, was associated with the spontaneous development of mammary intraepithelial neoplasia and invasive mammary carcinomas in a significant proportion of aged females. These data indicate that enforced mTERT expression can promote the development of spontaneous cancers even in the setting of ample telomere reserve.

Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility.

Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1(tm2Ejn), referred to as Delta C) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Delta C(-/-)) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and Delta C(-/-) MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1(tm2Ejn/tm2Ejn) mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions.

Genetic analysis of Pten and Ink4a/Arf interactions in the suppression of tumorigenesis in mice.

Dual inactivation of PTEN and INK4a/ARF tumor suppressor genes is a common feature observed in a broad spectrum of human cancer types. To validate functional collaboration between these genes in tumor suppression, we examined the biological consequences of Pten and/or Ink4a/Arf deficiency in cells and mice. Relative to single mutant controls, Ink4a/Arf-/-Pten+/- mouse embryonic fibroblast cultures exhibited faster rates of growth in reduced serum, grew to higher saturation densities, produced more colonies upon low density seeding, and showed increased susceptibility to transformation by oncogenic H-Ras. Ink4a/Arf deficiency reduced tumor-free survival and shortened the latency of neoplasias associated with Pten heterozygosity, specifically pheochromocytoma, prostatic intraepithelial neoplasia, and endometrial hyperplasia. Compound mutant mice also exhibited an expanded spectrum of tumor types including melanoma and squamous cell carcinoma. Functional synergy between Ink4a/Arf and Pten manifested most prominently in the development of pheochromocytoma, prompting an analysis of genes and loci implicated in this rare human neoplasm. The classical pheochromocytoma genes Ret, Vhl, and Nf-1 remained intact, a finding consistent with the intersection of these genes with pathways engaged by Pten and Ink4a/Arf. Notably, conventional and array-comparative genomic hybridization revealed frequent loss of distal mouse chromosome 4 in a region syntenic to human chromosome 1p that is implicated in human pheochromocytoma. This study provides genetic evidence of collaboration between Pten and Ink4a/Arf in constraining the growth and oncogenic transformation of cultured cells and in suppressing a wide spectrum of tumors in vivo.

Restoration of Transforming Growth Factor-β Signaling Enhances Radiosensitivity by Altering the Bcl-2/Bax Ratio in the p53 Mutant Pancreatic Cancer Cell Line MIA PaCa-2

In this study, we investigated whether lack of transforming growth factor beta (TGF-beta) type II receptor (RII) expression and loss of TGF-beta signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-beta-responsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing antibodies to TGF-beta, indicating that these changes were dependent on TGF-beta signaling. Compared with MIA PaCa-2/vector cells, MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x(L) or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-beta signaling-mediated apoptosis. Thus, restoration of TGF-beta signaling sensitized these cells to ionizing radiation, although these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-beta signaling and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important component of radiation-induced TGF-beta signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a ( Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a −/− cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a +/+ and Gadd45a −/− cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Oncogenic and mutagenic effects of UV in mammalian cells

Ultraviolet light is present in the solar system and can cause major biological effects. The potential cytotoxic, mutagenic, and carcinogenic effects of UV have been studied at cellular and molecular level. Using cultured mouse embryonic fibroblasts (C3H10T1/2), we investigated the induction of mutation and transformation by UV and/or X-rays. Studies were also done with normal human mammary epithelial cells for cell inactivation and mutation induction. Curvlinear dose-response curves were observed for mutation and oncogenic transformation. The interaction between UV and X-rays depends on the sequence of exposure. When UV was given following X-irradiation, there was an additive effect. When UV was given prior to X-irradiation, however, there was a synergistic effect for both cell inactivation and transformation. The basic lesion(s) important for somatic mutation and transformation remains to be determined, and the fundamental mechanism(s) of UV and ionizing radiation interaction remains to be elucidated.

Production and identification of natural monocyte chemotactic protein from virally infected murine fibroblasts

Primary cultures of mouse embryonic fibroblasts and confluent monolayers of mouse fibroblastoid cells (L929) were found to secrete a chemotactic factor specific for monocytes. It biological activity was deduced from both the migration distance under agarose and the number of migrated monocytes in the micropore filter method. The monocyte chemotactic protein (MCP) was inducible in these cells by double-stranded RNA and by infection with virus. In embryonic fibroblasts MCP was also produced in response to the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Under all conditions for induction of MCP tested no production of chemotactic activity for granulocytes could be detected. MCP activity from virally infected L929 cells was concentrated and purified by sequential adsorption to controlled pore glass, heparin-Sepharose chromatography, ion-exchange FPLC and reversed-phase HPLC. Pure MCP was found to occur mainly as a 7-8-kDa protein. Although the mature protein possessed a blocked NH2-terminus, it was identified by enzymatic cleavage and sequence analysis of an internal fragment. The sequence obtained corresponded to a part of the cDNA-derived protein sequence of the murine 'competence' (JE) gene, inducible in fibroblasts by cytokines and virus. In all probability the 7-8-kDa MCP form represents the natural product of the mouse gene JE. Murine MCP can thus be classified in the novel family of small inducible inflammatory proteins.

An Interferon-Induced 16-kD Protein Is Present in the Membranes of Interferon-Sensitive but Not in Interferon-Resistant Mouse Cells

Human interferon-alpha (IFN-alpha) or IFN-beta has been shown to induce a 17-kD membrane protein in human cells which when eluted from SDS gels inhibited the multiplication of cells of different human cell lines. We show herein that mouse IFN-alpha/beta induces a 16-kD membrane protein in L1210 and Friend erythroleukemia cells sensitive to IFN-alpha/beta, (but not in the derived IFN-alpha/beta-resistant cell lines) as well in primary and monolayer cultures of mouse embryonic fibroblasts and adult mouse hepatocytes, and in suspensions of spleen cells. In addition, IFN-alpha/beta enhanced the expression of an 11-kD membrane protein which could be shown by immunoprecipitation to be beta 2-microglobulin. Anticell proliferation activity was not recovered from the 16-kD fraction of the SDS gels.

Effect of the double-stranded polynucleotide complex polyadenylate-polyuridylate (Poly A-U) on interleukin-6 production by mouse fibroblasts

Cultures of mouse embryonic fibroblasts (L 929) have been shown to produce a factor which promotes the growth of B cell hybridoma (hybridoma growth factor, HGF) i.e, interleukin 6 (IL-6). The aim of the present study was to investigate the effect of Poly A-U on IL-6 production by this cell type. After incubation for 48 h at 37°C of confluent (1 week old) L 929 fibroblasts in the presence or in the absence of Poly A-U, IL-6-like activity in supernatants was measured by the proliferation assay of the IL-6-dependent B cell hybridoma cell line, 7TD1. Poly A-U increased IL-6 activity in supernatants in a dose-dependent manner at doses higher than 50 μg/ml, the maximum activity being observed at the highest concentration of Poly A-U used, i.e. 500 μg/ml. β Interleukin-1 (β IL-1) and poly-cytidylic-polyinosinic (Poly I-C) have been shown to be inducers of IL-6 in fibroblast culture and thus their effect was compared to that of Poly A-U. The IL-6 activity in supernatants induced by 500 μg/ml Poly I-C (58.4 ± 16.4 U/ml; n = 4) was higher than that evoked by 100 U/ml β IL-1 (5.7 ± 0.4 U/ml) or 500 μg/ml Poly A-U (39.6 ± 7.8 U/ml). The increased production of IL-6 by Poly A-U may explain part of its previously re[orted immunomodulatory effects.

Diphenylhydantoin and mitotic spindle abnormalities in cultured mouse and human cells

The present study demonstrates that the antiepileptic drug diphenylhydantoin (DPH) is capable of inducing aneuploidy but not structural aberrations in cultured mouse embryonic fibroblasts. A high concentration of 200 μg/ml was found to increase the percentage of hyperdiploidy from 4.8 (control) to 16.0. The treatment was found to increase mitotic indices as a consequence of a mitotic-arresting action of the drug. These effects are probably due to the effect of the drug on the structure of the mitotic apparatus. Abnormal cell divisions and mitotic disturbance were found to increase in a dose-dependent manner after DPH treatment. In a parallel study, human amnion cells were found to show similar response to DPH treatment.

Role of the cardiopulmonary blood volume in changes in left ventricular output during stimulation of somatic afferent nerve fibers

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.

Fibronectin localization and endocytosis in early and late mouse embryonic fibroblasts in primary culture: A study by light and electron microscopic immunocytochemistry

In spreading fibroblasts, strong endocytosis of exogenous fluorescent fibronectin (FN) was observed from the beginning of their attachment to the substratum. In early fibroblasts, the internalized FN was localized both in the peripheral ruffles and in the perinuclear cytoplasm, in late fibroblasts, whose spreading was slower, FN uptake was not detected in the ruffles. In growing cultures, supracellular FN fibres, detected by direct fluorescence microscopy or by the indirect peroxidase-anti-peroxidase (PAP) complex technique, were scarce on early cells, but very numerous on the upper face and on the filopodia of late cells. At the ultrastructural level, FN, localized with the immuno-gold staining method, was found associated with fibres of the extracellular matrix and the upper face of the cells. FN was endocytosed via smooth vesicles and we suggest that the internalization process is slower in the late cells. In confluent early cultures, an extended network of pericellular FN was observed as usual. The pericellular FN of late grouped cells was present as a few coarse fibres connected with some of the cell surface threads.

Actin content and organization of microfilaments in primary cultures of mouse embryonic fibroblasts (in vitro ageing).

Actin distribution in serially passaged embryonic mouse fibroblasts has been visualized by the anti-actin-PAP method; the organization of the microfilaments has been observed by electron microscopy (SEM and TEM). Four successive actin patterns have been identified: early (few well-organized bundles of microfilaments), middle-aged (many well-organized bundles and patches around the nucleus), late (numerous ill-organized filamentous structures and diffuse perinuclear-actin) and "senescent" (heavy packs of short microfilaments around the nucleus). All the observed actin-positive filaments were disrupted by cytochalasin B treatment. The cytoplasmic actin complex was cell-age and not cell-size-dependent; it behaved differently from the cytoplasmic microtubular complex to serially subcultivated fibroblasts. Measurements of the cell-protein content (Lowry's method) and SDS-polyacrylamide gel electrophoresis (Laemmli's method) have been performed in the successive population doubling levels (PDL) of the primary cultures. Triton-insoluble actin increased in parallel with total protein and reached about 4% of the total proteins in all the PDLs. Triton-soluble actin also increase at the beginning of the middle-aged period (generally 6 PDL) and another in declining cultures (generally 10 PDL). Total actin amounted to about 8% of the total proteins in early fibroblasts, to about 16% at the beginning of the middle-aged period and to about 20% in the declining terminal cultures. Taking into account all the known characteristics of subcultivated primary cultures, we tentatively consider the evolution of the fibroblasts as an in vitro differentiation followed by true in vitro senescence in the declining cultures. Regarding the cytoplasmic actin-complex, senescence would be characterized by a sharp increase in soluble actin, an unbalanced ratio between soluble and insoluble actin and an impairment of the ability of the microfilaments to form well-organized bundles.

Membrane expression of Fc-receptors in cultured leukemic cell lines. I. Induction of Fc-receptor in undifferentiated types of cells after passive modulation of lipid viscosity.

The role of lipid environment of plasma membrane for the expression of Fc receptor (gamma) (FcR gamma) were studied by physicochemical modification of the membranes of myeloid leukemia cell line cells, M1. Sterol binding polyene antibiotics such as amphotericine B or filipin inhibit the expression of FcR gamma during the differentiation of M1- cells by the CM obtained from the culture of mouse embryonic fibroblasts, suggesting the possible involvement of cholesterol molecules in the induction mechanism of FcR gamma. Low-temperature incubation at either 20 degrees C or 4 degrees C results in the increment of their membrane microviscosity, when tested by the fluorescence depolarization method, in company with the appearance of FcR gamma on approximately 60% of M1 cells when assayed by the EA-rosetting method. FcR gamma is also induced on approximately 40% of M1- cells by incubating cells with cholesterol-phosphatidyl-choline liposomes with the concomitant increase of their membrane microviscosity. Nevertheless, no phagocytosis of EA is observed in these cells treated with such physicochemical procedures. These findings imply that FcR gamma is embedded in the membrane of M1- cells and that physicochemical modification of the membrane lipid bilayers can induce the FcR gamma independent on their functional differentiation marked by the expression of phagocytic activity.

Metabolism of steroids by mouse teratocarcinoma cells

Steroid metabolism was studied in cells of a pluripotent mouse teratocarcinoma (TC) line OC15S1. One to five day old monolayer cultures were incubated for 3 to 20h in the presence of radioactive pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione or estradiol-17β. Extracted radioactivity was analyzed by thin layer chromatography, gas liquid chromatography and recrystallizations to constant specific activity and constant isotope ratio. The substrates were metabolized to different extents and most of the metabolites were excreted into the medium. The age of the culture did not affect the results. Similar types of metabolic conversion of steroids was obtained by cultured mouse embryonic fibroblasts which were used as a reference. The results indicated that the cultured TC cells are not capable of 1. efficient conversion of pregnenolone to progesterone nor of 2. significant modification of the major exogenous estrogen, estradiol-17β, but are capable of 3. efficient modification of progesterone and of 4. converting pregnenolone to a more polar form, as well as of 5. metabolizing both DHA and androstenedione.

Inhibition of cell growth by near ultraviolet light photoproducts of tryptophan.

Exposure of dilute aqueous solutions of tryptophan to near UV light (320 to 390 nm) at subsolar levels yields fluorescent photoproducts capable of inhibiting the growth and differentiation of cultured mouse embryonic fibroblasts and fertilized sea urchin eggs. The ability of these cells to incorporate labelled precursors of protein, RNA, and DNA into their respective macromolecules was markedly inhibited by adding tryptophan preirradiated with near UV light to their incubation media. Thus the inhibition of growth and differentiation of these cells seems to result from a depression of their ability to synthesize macromolecules in the presence of the photoproducts.

Effect of colcemid on cell migration, mitosis, and DNA synthesis in mouse embryonic fibroblast cultures

Effect of colcemid on cell migration, mitosis, and DNA synthesis in mouse embryonic fibroblast cultures

Glucocorticoids and cellular immunity in vitro. Facilitation of the sensitization phase and inhibition of the effector phase of a lymphocyte anti-fibroblast reaction.

We studied the influence of glucocorticoids on the sensitization phase as well as on the cytolytic effector phase of an in vitro lymphocyte-mediated immune reaction. Lymphocytes obtained from the spleens or lymph nodes of unimmunized inbred rats were sensitized against foreign rat or mouse embryonic fibroblasts in cell culture. The capacity of the sensitized lymphocytes to produce a cytolytic effect was tested by transferring them to target fibroblast cultures. Injury to target fibroblasts was measured by release of radioactive (51)Cr from previously labeled fibroblasts or by direct count of viable fibroblasts after incubation with sensitized lymphocytes. Various concentrations of water-soluble hydrocortisone or prednisolone were added to cell cultures during the 5 day sensitization phase and/or during the subsequent cytolytic effector phase and the influence of these hormones on the number and cytolytic capacity of the lymphocytes was measured. During the sensitization phase, the presence of glucocorticoid hormones, at concentrations of about 1 microg/ml, led to a profound decrease in the total number of recoverable lymphocytes. However, the per cent of large transformed lymphocytes was much greater in these treated cultures. The antigen-specific cytolytic capacity per cell of the glucocorticoid-treated lymphocytes, after the hormone was removed, was several times greater than that of lymphocytes sensitized in the absence of added hormones. Glucocorticoids influenced the effector phase of the reaction by inhibiting lymphocyte-mediated injury to target fibroblasts. The hormones, at concentrations of about 1 microg/ml, inhibited the cytolytic effect by about 50% without reducing the viability of the sensitized lymphocytes. Dose-dependent toxicity to lymphocytes and increasing inhibition of cytolytic effect appeared at higher concentrations of hormones. Thus, hydrocortisone and prednisolone, at concentrations of about 1 microg/ml, did not suppress the induction of sensitization, a process which they seem to facilitate in vitro. However, similar concentrations of these hormones appear to inhibit the cytolytic effector mechanism of sensitized lymphocytes. These findings may be relevant to the use of glucocorticoids as immunosuppressive agents in vivo.

Electrical coupling: low resistance junctions between mitotic and interphase fibroblasts in tissue culture.

Dividing cells of chick embryonic fibroblasts and of mouse embryonic fibroblasts (3T3) in tissue culture are electrically coupled to their interphase neighbors. Recordings from many such cells suggest that this coupling persists throughout the division cycle of the mitotic cell.