Rat Embryo Fibroblast Cell Line Research Articles

Synthesis of 5‐Fluorouracil–Naproxen Conjugates as a Mutual Prodrug for Targeting Cancer Tissues

AbstractA new 5‐fluorouracil–naproxen conjugate is synthesized as a mutual prodrug for targeting cancer tissues. The structure of the target compound and their intermediate are characterized by their melting point, IR, 1H NMR, 13C NMR, and elemental microanalysis. The cytotoxic activity is preliminarily evaluated using nonsmall lung cancer CRL‐2049, human breast cancer CAL‐51, and one type of normal cell line; rat embryo fibroblast cell line. The synthesized compound shows a good cytotoxic effect at the cancer cell and no significant effect at rat embryo fibroblast cell line.

Cyperus rotundus L. alkaloid extracts enhance oncolytic Newcastle disease virus against digestive system neoplasms

Digestive system cancers are common primary cancer in humans. Plants are an excellent source of compounds used in the treatment of different types of cancer. Virotherapy is a promising tool for fighting cancer because of its safety and selectivity. Thus, the present study proposed a new combination therapy consisting of secondary metabolite compounds (alkaloids) extracted from Cyperus rotundus L. and oncolytic Newcastle disease virus (NDV). The new combination was tested against three cancer cell lines (i.e., human rectal and esophagus cancer and mouse hepatocellular carcinoma) and a non-transformed rat embryo fibroblast cell line (REF). The MTT viability assay was used to analyze the possible synergism using the Chou–Talalay analysis method. The in vitro study showed that the combination therapy of NDV-alkaloid extract had synergistic enhanced anticancer activity on all the cancer cell lines tested. Moreover, combination therapy showed antagonism and low cytotoxic effect on the REF cell line. NDV-alkaloid treatment was found to upregulate p53 in comparison with each monotherapy alone, and thereby it is a possible explanation for the synergism. In conclusion, this study proposed a novel cancer therapy via the combination of C. rotundus L. (alkaloids) extract and oncolytic NDV, which may be a promising therapy for gastrointestinal tumors.

Study The Effect of Purifed Goat Milk Lactoferrin on HeLa Cancer Cell line Growth In vitro

Lactoferrin is an important protein in many biological applications as a potential cancer treatment agent .In this study, lactoferrin was purifed from goat colostrum by Ion exchange chromatography by using CM-Sephadex G-150 column and gelfltration by using Sephadex G-200 column .To evaluted its ability as anticancer agent the study utilized an in vitro evaluation for the cytotoxic effect of the purifed goat milk lactoferrin (gLf) on two cell lines, HeLa (Human cervical cancer) cell line and Rat Embryo Fibroblast (REF) cell line at different concentrations and different exposure time of treatment. The purifed gLf concentrations ranging (19.53 to 5000) μg/ml for 24,48 and 72 hours. The effect of gLf was evaluated by employing MTT assay. The results revealed signifcant cytotoxic effect at levels (P<0.05) for all concentrations and for all exposure time of gLF on HeLa cell line as compared to untreated control cells, The inhibition rate IR% increased with raising of gLF concentration and incubation period The highest inhibitory growth was at the concentration ( 5000 µg/ml) after 72hrs of exposure time (64.38%) ,while only the highest concentration gave signifcant inhibitory effect (P< 0.05) with normal cell REF .

Study of cytotoxicity of purified toxin A produced from pseudomonas aeruginosa on cell lines

Pseudomonas aeruginosa is the leading etiology of gram negative bacteria causing diseases at most medical centers . The bacterium’s virulence depends on number of cell-associated and extracellular factors. Exotoxin A among these virulence factors are toxic for a wide variety of cell line, it inhibits protein synthesis in these lines as well as in numerous organs in animals models. In vitro cytotoxic effects of purified toxin A on two cell lines Rat embryo fibroblast cell line (REF) and on tumor cell line Rhabdomyosarcoma cell line (RD) were studied. Results revealed that toxin A had significant cytotoxic effects on both cell lines. Conclusion: In vitro evaluating the cytotoxic effects of purified toxin A produced from pseudomonas aeruginosa at different concentration on normal cell line Rat embryo fibroblast cell line (REF) ) and on tumor cell line Rhabdomyosarcoma cell line (RD) revealed that purified exotoxin A has significant inhibitory effects on REF cell line which was dose and time dependent. On the other hand cytotoxic effects of purified toxin A on tumor cell line (RD) reveal that inhibition in cell viability was dependent on concentration of purified toxin A but independent on time. Key words : Toxin A, cytotoxicity assay, cell line

Cytotoxicity of purified methionine γ- lyase produced by Pseudomonas putida on several cell lines

Many human malignant cell lines and primary tumours have absolute requirements for L-methionine (1). Under normal circumstances methionine comes from dietary proteins, most normal tissues can also synthesize methionine from either homocysteine or methylthioadenosine (2). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and became arrested in the late S/ G2 phase of the cell cycle (3).Methionine cleaving enzyme (methionine γ- lyase) has been found to be an effective antitumour agent in vitro as well as in vivo (4). Methionine γ- lyase is a pyridoxal 5-phosphate dependent enzyme that catalyzes the α, γ- elimination of L-methionine to α-ketobutyrate, methanethiol and ammonia (5). Rhabdomyosarcoma (RD) cell line at passage 65, AhmedMohammed-Nahi-2003(AMN3) cell line at passage 180, AMGM (Ahmed – Majeed-Glioblastoma-Multiform) cell line at passage 65 and normal Rat Embryo Fibroblast (REF) cell line at passage 56, kindly provided by Iraqi center for cancer and medical genetics research (ICCMGR) were cultured in PRMI 1640 medium supplement with 10% heat inactivated fetal calf serum and incubated at 37 ºC with 5% CO2. Examination of cytotoxicity of methionine γ- lyase Cytotoxicity was determined with the crystal violet stain as previously described (7). In briefly , 1x105 cell/ml was seeded onto 96-well culture plates in 200 µl RPMI 1640 medium and incubated until the cell reached confluent monolayer(vary according to the kind of cell-line). After incubation period the medium was removed and 200 μl of various concentrations (1000, 500,250 and 125 μg/ml) from methionine γ- lyase were added to the plate by using 3- well replicates for each concentration. The plates were incubated at 37ºC for 96 hr. The control (cancer cell lines without treatment with enzyme) (3-well replicates) was treated with 200 μl of serum free medium and incubated at 37ºC for 96 hr. At the end of incubation period, the enzyme and medium was removed from plate and washed with PBS to remove unattached (dead) cells. Two hundred μl of crystal violet was added to each well and left for 20 min at 37ºC.The stain was removed by washing with tap water several times, and the plates were left to dry. After drying, each plate was read by using ELISA microplate spectrophotometer at 492nm wave length. The percentages of Inhibitory Rate (IR %) were estimated (8).

The Cytotoxic Activity of Punica granatum on Growth of Hela and REF Cell Lines

The crude aqeouse extracts of punica granatum peel were assessed for their cytotoxic activity assay.This study utilized two different types of cell lines, Hela (Cervical human carcinoma) and REF (Rat Embryofibroblast).The results showed that the aqueous extract of punica granatum peel exhibited high cytotoxicity against theREF cell line with an inhibition value of 97.3 μg/ml after 24 h incubation. However, punica granatum peeldisplayed less toxicity against the Hela cell line with an inhibition value 24.4 μg/ml after 24 h incubation andthis effect was dose- and time-dependent.In conclusion, the results demonstrated that punica granatum peel is potently cytotoxic against REF cell,and lesser extend against Hela cells.

The in vitro assessment of dipyridophenazine complexes in H-ras oncogene transformed rat embryo fibroblast 5RP7 cell line.

Purpose The aim of this study is to detect apoptotic and cytotoxic/antiproliferative effects of a ligand substance and its metal derivatives. The substances were investigated by using an h-ras oncogene transformed rat embryo fibroblast cell line (5RP7). Methods The cytotoxic influences of dipyrido[3,2-a:2',3'c]phenazine ligand, dipyrido[3,2-a:2',3'c] phenazine-platinum(II) complex ([Pt(dppz)Cl2]) and dipyrido[3,2-a:2',3'c] phenazine-gold(III) complex ([Au(dppz)Cl2]Cl) were determined with MTT (3[4,5-dimetiltiyazol2-yl]-2,5-difeniltetrazolyum bromid) assay on 5RP7 cells. Results Dipyrido[3,2-a:2',3'c] phenazine, dipyrido[3,2-a:2',3'c] phenazine-platinum(II) complex ([Pt(dppz)Cl2]) and dipyrido[3,2-a:2',3'c] phenazine-gold(III) complexes ([Au(dppz)Cl2]Cl) caused significant increase in cytotoxicity in a dose and time dependent manner. The effects of dipyridophenazine ligand (dppz) and its metal derivatives on apoptosis were monitorized using cytotoxic dose (10μM) DAPI fluorescent staining. It was shown that dppz and its compounds induced apoptosis. Conclusions These findings show that dpzz and its complexes can be studied as novel alternative chemotherapeutics in cancer treatment.

In Vitro Cytotoxic Study for Purified Resveratrol Extracted from Grape Skin Fruit Vitis vinifera


 This study was conducted with the aim to extract and purify a polyphenolic compound “ Resveratrol” from the skin of black grapes Vitis vinifera cultivated in Iraq. The purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis with 10% HCl solution then the aglycon moiety was taken with organic solvent
 ( chloroform). Using silica gel G60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid 20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). The study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma AMN-3 cell line, the human laryngeal carcinoma (Hep-2) cell line and the Rat embryo fibroblast (Ref) cell line, at different concentrations and different expousure time. The cytotoxic effect of the pure resveratrol was studied in comparison with trans- resveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations (0.05 , 0.1 , 0.2 and 0.4) µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. The cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (P<0.01) was achieved after 24 hr of exposure on both AMN-3 and Ref cell lines. Hep-2 cell line responded to extracted resveratrol in different manners.
 Keywords: Resveratrol , grape skin , polyphenols, cytotoxic study

The antiapoptotic gene bcl-2 prevents reactivation of the senescence program induced by the histone deacetylase inhibitor sodium butyrate in rat fibroblasts transformed by the oncogenes E1A and c-Ha-Ras

The role of the gene bcl-2 conferring resistance to apoptosis in the activation of the cellular senescence program induced by the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBut) has been studied in transformed rat fibroblasts. The work was carried out in the rat embryo fibroblast cell line transformed by the oncogenes E1A, cHa-Ras, and bcl-2 (ERasBcl cells) resistant to apoptosis. The parent line of E1A-and cHa-Ras-transformed cells (ERas cells) was used as a control. It has been shown that NaBut suppresses the proliferation of ERasBcl cells to a much lesser extent than do the control ERas cells, in spite of the delay in cell cycle progression in the G1 phase for both lines. NaBut-induced hypertrophy is less marked in ERasBcl cells than in ERas cells due to the lower level of activation of the mTORC1 complex controlling protein synthesis and ribosome biogenesis. The activation of mTORC1 was assessed from the phosphorylation of its targets: ribosomal protein S6 and the inhibitor of translation initiation factor eIF4E (4E-BP1). Investigation of the dynamics of the level of one of the major markers of autophagosome formation (protein LC3) has shown the transient character of NaBut-induced accumulation of lipid-bound form LC3-II marking autophagosome membranes in bcl-2-expressing transformants in contrast to the ERas line. In addition, in ERasBcl cells there was no activation of the senescence-associated β-galactosidase (the marker for senescent cells). The results obtained suggest that the high level of expression of bcl-2, which blocks apoptotic cell death, prevents the implementation of the NaBut-induced tumor-suppressive cellular senescence program.

Mitochondrial Activity of the Locally Established Rat Embryo Fibroblast Cell Line Through Different Passages

Background: Mitochondria are the cell’s powerhouse, the site where the vast majority of ATP is synthesized. Mitochondrial activity represent a central checkpoint for detection of the difference between cancer cells and normal cells at the metabolic profile.Objective: To find out if there is a correlation between in vitro transformation and mitochondrial activity, by measuring the activity during clonal evolution of the locally established rat embryo fibroblast (REF) cell line throughout studying different passages.Materials & Methods: The (REF) cell line, was in vitro cultured. Mitochondria were isolated by differential centrifugation following Mitochondria Isolation Kit. Enzymatic activity of intact mitochondria has been measured using Cytochrome C Oxidase Activity Assay Kit. The decrease in absorbance at 550nm as horse heart ferrocytochrome c was oxidized has been monitored.Results: Depending on the conducted colorimetric assay, cytochrome c oxidase activity was measured, for the three different passages (42, 72 and 91) of REF cell line. At 550 nm, spectral data were valued (0.247, 0.723 and 1.318) unit/ml for three passages of REF cells (42, 72 and 91) respectively.Conclusion: There was a significant relationship between the mitochondrial activity and the age (passage number) for these in vitro cultured cells. These variations probably due to the transformational events that have been occurred during long-term continuous subculture

Initial Characterization of a Low-Molecular-Weight Factor Enhancing the Checkpoint Response

In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

In vitro cytotoxic study for partially purified resveratrol extracted from grape skin fruit Vitis vinifera

The partial purified resveratrol was obtained from the skin of black grape fruit cultivated in Iraq using 80% ethanolic solution, then an acid hydrolysis with 10% HCl solution for (10–30) min at 60Cº was carried out. The aglycone moiety was taken with an organic solvent (chloroform), then using an open glass column packed with silica gelG 60 as a stationary phase and a mobile phase of; benzene: methanol: actic acid (20:4:1). The study utilized an in vitro evaluation for the cytotoxic effect of the partially purified resveratrol on some cell lines including, the murine mammary adenocarcinoma (Ahmed –Mohammed –Nahi–2003 -AMN -3) cell line; the human laryngeal carcinoma (Hep -2) cell line and the Rat Embryo Fibroblast (REF) cell line at different concentrations and different exposure time of treatment. The partial purified resveratrol extract concentrations ranging (7.8–4000) µg/ml in a two fold serial dilutions were used to treat the three types of cell lines for 48 and 72 hours intervals. AMN-3 cell lines showed highest sensitivity toward the cytotoxic effect of the paritial purified resveratrol than other cell lines after 48 hours in a dose dependent manner. While Hep-2 cell line showed novel behavior, the lowest concentration of cell treatment gave the most significant (P< 0.01) inhibitory effect. Only the highest concentration gave significant inhibitory effect (P< 0.01) with the transformed Ref cell line.

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts.

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.

Identification of novel candidates for replicative senescence by functional proteomics.

To identify the underlying mechanisms that limit the mitotic potential of normal somatic cells, we have undertaken a high resolution differential proteomic analysis aimed at identifying proteins that were differentially expressed upon replicative senescence. Since replicative senescence in heterogeneous primary fibroblast cultures is asynchronous, we analysed a group of conditionally immortalized rat embryo fibroblast cell lines that have previously been shown to undergo synchronous senescence upon inactivation of SV40 tsA58 T antigen. This identified 43 spots that were differentially expressed in these cell lines. Comparison of the identity of these features with those identified in a complimentary independent differential proteomic analysis of replicative senescence, directly in primary rat embryo fibroblasts upon serial passaging, identified nine features that were in common between the two studies even though they had been conducted entirely separately. None of these proteins have previously been recognized to be involved with replicative senescence. Thus, they represent novel starting points for elucidating the underlying mechanism that regulates the finite mitotic life span of somatic cells and how it can be overcome in cancer cells.

βIII Spectrin Binds to the Arp1 Subunit of Dynactin

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

The Radioresistance to Killing of A1–5 Cells Derives from Activation of the Chk1 Pathway

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

Metabolic Imaging in Multicellular Spheroids of Oncogene-transfected Fibroblasts

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.

DNA as an important target in radiation-induced apoptosis of MYC and MYC plus RAS transfected rat embryo fibroblasts

Purpose : This study uses a radiation chemistry approach to determine if DNA is an important target for radiation-induced apoptosis of myc (MR4) and myc plus ras (3.7) transfected rat embryo fibroblast cell lines. Materials and methods : The radiation protection efficiency of four thiols was compared with net molecular charge ranging from -1 to +2: mercaptopropionic acid (Z = -1), mercaptoethanol (Z = 0), cysteamine (Z = +1), N(2-mercaptoethyl)-1,3-diaminopropane (Z = +2). Protection factors were determined for these thiols against radiation-induced apoptosis (Apoalert assay), mitotic cell death (clonogenic assay) and double-strand break (dsb) induction (pulse field gel electrophoresis) in MR4 and 3.7 cells. Theoretical protection factors for these thiols against dsb induction were also calculated from second-order chemical repair constants for single-strand breaks (ssb) and the concentration of added thiols in MR4 and 3.7 cell lines. Results : The charge-dependent increases observed for measured protection factors against radiation-induced apoptosis did not differ significantly between the two cell lines, nor did they differ significantly from the corresponding increases observed for radiation-induced mitotic cell killing and for induction of dsb. The calculated protection factor for dsb also showed a thiol charge-dependent increase similar to the measured protection factors for all of the other parameters studied. Conclusions : These results are consistent with the hypothesis that DNA is an important target for radiation-induced apoptosis.

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53.

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.

P53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation.

The proliferating cell nuclear antigen (PCNA) is a highly conserved cellular protein that functions both in DNA replication and in DNA repair. Exposure of a rat embryo fibroblast cell line (CREF cells) to gamma radiation induced simultaneous expression of PCNA with the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). PCNA mRNA levels transiently increased in serum-starved cells exposed to ionizing radiation, an observation suggesting that the radiation-associated increase in PCNA expression could be dissociated from cell cycle progression. Irradiation of CREF cells activated a transiently expressed PCNA promoter chloramphenicol acetyltransferase construct through p53 binding sequences via a mechanism blocked by a dominant negative mutant p53. Electrophoretic mobility shift assays with nuclear extracts prepared from irradiated CREF cells produced four p53-specific DNA-protein complexes with the PCNA p53 binding site. Addition of monoclonal antibody PAb421 (p53-specific) or AC238 (specific to the transcriptional coactivator p300/CREB binding protein) to the mobility shift assay distinguished different forms of p53 that changed in relative abundance with time after irradiation. These findings suggest a complex cellular response to DNA damage in which p53 transiently activates expression of PCNA for the purpose of limited DNA repair. In a population of nongrowing cells with diminished PCNA levels, this pathway may be crucial to survival following DNA damage.