Cloned Rat Embryo Fibroblast Research Articles

Astrocyte elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis

Astrocyte-elevated gene-1 (AEG-1) expression is increased in multiple cancers and plays a central role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Additionally, overexpression of AEG-1 protects primary and transformed human and rat cells from serum starvation-induced apoptosis through activation of PI3K/Akt signaling. These findings suggest, but do not prove, that AEG-1 may function as an oncogene. We now provide definitive evidence that AEG-1 is indeed a transforming oncogene and show that stable expression of AEG-1 in normal immortal cloned rat embryo fibroblast (CREF) cells induces morphological transformation and enhances invasion and anchorage-independent growth in soft agar, two fundamental biological events associated with cellular transformation. Additionally, AEG-1-expressing CREF clones form aggressive tumors in nude mice. Immunohistochemistry analysis of tumor sections demonstrates that AEG-1-expressing tumors have increased microvessel density throughout the entire tumor sections. Overexpression of AEG-1 increases expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2, and hypoxia-inducible factor 1-alpha. In vitro angiogenesis studies further demonstrate that AEG-1 promotes tube formation in Matrigel and increases invasion of human umbilical vein endothelial cells via the PI3K/Akt signaling pathway. Tube formation induced by AEG-1 correlates with increased expression of angiogenesis markers, including Tie2 and hypoxia-inducible factor-alpha, and blocking AEG-1-induced Tie2 with Tie2 siRNA significantly inhibits AEG-1-induced tube formation in Matrigel. Overall, our findings demonstrate that aberrant AEG-1 expression plays a dominant positive role in regulating oncogenic transformation and angiogenesis. These findings suggest that AEG-1 may provide a viable target for directly suppressing the cancer phenotype.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Progression elevated gene-3, PEG-3, induces genomic instability in rodent and human tumor cells.

Genomic instability is a fundamental component of cancer progression. Subtraction hybridization identified a novel rodent gene, progression elevated gene-3 (PEG-3) whose expression directly correlates with cancer aggressiveness and progression. Moreover, ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed phenotype. We demonstrate that PEG-3 expression in rodent tumor cells correlates directly with genomic instability as characterized by alterations in chromosome composition and structure. Additionally, elevated endogenous or ectopic expression of PEG-3 in rodent and human tumor cells, respectively, enhances gene amplification, as monitored by resistance to methothrexate (MTX) and amplification of the dihydrofolate reductase (dhfr) gene. Stable expression of PEG-3 in normal cloned rat embryo fibroblast (CREF) cells marginally elevates MTX resistance, but morphology remains unaltered and anchorage independence is not induced, suggesting that these phenotypes are separable in immortal cells and gene amplification may precede the acquisition of morphological and oncogenic transformation. The present studies document that stable, inducible, and transient expression of PEG-3 in cancer cells augments genomic instability. In these contexts, one mechanism by which PEG-3 influences cancer progression may be by preferentially facilitating the development of genomic changes in evolving cancer cells.

Dual action of the adenovirus E1A 243R oncoprotein on the human proliferating cell nuclear antigen promoter: repression of transcriptional activation by p53.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

Caffeic acid phenethyl ester profoundly modifies protein synthesis profile in type 5 adenovirus-transformed cloned rat embryo fibroblast cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis from bee hives, exerts a plethora of biological changes in diverse systems. These include antimitogenic, anticarcinogenic, anti-inflammatory and immunomodulatory responses. CAPE directly induces programmed cell death (apoptosis) in type 5 adenovirus (Ad5)-transformed cloned rat embryo fibroblast cells, wt3A. To identify the gene and protein expression changes induced by CAPE in wt3A cells we used a strategy involving in vitro translation of mRNAs followed by high resolution two-dimensional (2D) gel electrophoresis. This approach results in the detection of 745 spots, including 172 displaying differences in expression upon exposure to CAPE. A high proportion of spots show profound changes in spot intensity (42 spots with increased and 27 spots with decreased intensity) following CAPE treatment. These studies provide a basis for comparing these changes to known protein patterns of various cell populations with an ultimate aim of identifying families of polypeptides responsible for the up- and down-regulation of cellular proteins during CAPE-induced apoptosis. Specific newly appearing or completely disappearing spots (52 and 51 molecular species, respectively) will be used to attempt to identify and retrieve their cDNA counterparts from an ordered cDNA library. These approaches represent a novel strategy for cloning genes associated with and potentially mediating apoptosis.

TRANSCRIPTIONAL SWITCHING MODEL FOR THE REGULATION OF TUMORIGENESIS AND METASTASIS BY THE HA-RAS ONCOGENE - TRANSCRIPTIONAL CHANGES IN THE HA-RAS TUMOR-SUPPRESSOR GENE LYSYL OXIDASE

A model system is described that allows an analysis of the molecular and biochemical changes associated with expression and suppression of the oncogenic and metastatic phenotype of cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed, anchorage-independent and both tumorigenic and metastatic in athymic nude mice and syngeneic Fischer rats. Co-expression of the Ha-ras oncogene and Krev-1 tumor suppressor gene in CREF cells results in suppression of in vitro transformation. In contrast, Ha-ras/Krev-1 transformed CREF cells retain, with greatly extended latency periods, both tumorigenic and metastatic capabilities in athymic nude mice. The present study investigates changes in the Ha-ms suppressor gene, rrg (lysyl oxidase), during expression and suppression of the oncogenic phenotype in CREF cells. Nontumorigenic CREF cells and CREF cells transformed by the Ha-ras and Krev-1 gene that express a suppression in in vitro transformation contain elevated levels of lysyl oxidase mRNA and protein. In contrast, Ha-ms and Ha-ras/Krev-1 nude mouse tumor- and nude mouse lung metastasis-derived CREF cells contain reduced levels of lysyl oxidase mRNA and protein. Nuclear run-on assays indicate that suppression of lysyl oxidase expression in transformed subclones of CREF cells correlates with a reduction in transcription of the lysyl oxidase gene. Taken together, the current studies support a transcriptional switching model in which lysyl oxidase expression correlates directly with suppression of the Ka-ms-induced transformation phenotype and escape from oncogenic suppression correlates with a transcriptional silencing of the lysyl oxidase gene and decreased lysyl oxidase mRNA and protein.

EXPRESSION OF THE TRANSFORMED PHENOTYPE INDUCED BY DIVERSE ACTING VIRAL ONCOGENES MEDIATES SENSITIVITY TO GROWTH SUPPRESSION INDUCED BY CAFFEIC ACID PHENETHYL ESTER (CAPE)

Caffeic acid phenethyl ester (CAPE) displays enhanced growth suppressive and toxic effects toward cloned rat embryo fibroblast (CREF) cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed CREF cells (Su et al, Mol Carcinogen 4: 231-242, 1991). The present study was conducted to determine if transformation of CREF cells with additional oncogenes rendered these cells sensitive to the antiproliferative effect of CAPE. Additionally, studies were conducted to determine if reversion of the transformed phenotype could modify CAPE sensitivity. CAPE displayed increased growth suppressive activity toward CREF cells transformed by a number of oncogenes, including Ha-ras, v-src, v-raf, human papillomavirus type 18 (HPV-18) and human papillomavirus type 51 (HPV-51). Employing Ha-ras-transformed CREF (Ha-ras) and Ha-ras-transformed CREF cells overexpressing the Krev-1 suppressor gene (Ha-ras/Krev-1), evidence for a direct relationship between expression of the transformed phenotype and CAPE sensitivity was demonstrated. Ha-ras/Krev-1 cells displaying a suppression of the transformed phenotype exhibited increased resistance to CAPE-induced growth suppression versus Ha-ras cells, whereas Ha-ras/Krev-1 cells escaping transformation-suppression following in vivo growth in nude mice displayed enhanced sensitivity to growth-suppression induced by CAPE. Similarly, mutant Ad5 (H5hr1)-transformed and v-src-transformed CREF cells displaying a stable reversion in transformation also displayed a reduced sensitivity to CAPE versus their transformed counterparts. These observations indicate a direct relationship between expression of the transformed phenotype and CAPE sensitivity. Elucidation of the mechanism by which CAPE selectively inhibits growth of transformed cells should provide important insights into the critical molecular changes mediating expression of the transformed state and could help identify cellular targets for cancer therapy.

Induction and progression of the transformed phenotype in cloned rat embryo fibroblast cells: studies employing type 5 adenovirus and wild-type and mutant Ha-ras oncogenes.

Transformation of cloned rat embryo fibroblast (CREF) cells with the wild-type 5 adenovirus (wtAd5) transforming genes E1A and E1B (which extend from 0 to 11.2 map units) results in morphologically transformed cells that exhibit an increased saturation density in monolayer culture and display an anchorage-independent phenotype. WtAd5-transformed CREF (wtAd5 CREF) cells do not, however, induce tumors when injected subcutaneously into athymic nude mice or syngeneic Fischer rats. We have analyzed the effect of the ras oncogene and site-specific mutants in the ras oncogene that result in p21 proteins with altered biochemical properties on the oncogenic and metastatic properties of singly (ras) and doubly (ras + wtAd5) transformed CREF cells. Transformants expressing the wild-type ras p21 protein and ras mutants producing p21 proteins that retained GTP-binding properties grew in agar, induced tumors in nude mice and syngeneic rats, and metastasized to the lungs of rats when injected into their tail veins. In contrast, cells transformed with the ras mutant 116K (which contains a mutation at residue 116 that produces a Lys instead of an Asn and does not bind GTP or induce CREF cells to grow in agar) did not become morphologically transformed and were not oncogenic when injected subcutaneously into either nude mice or Fischer rats; further, such cells were not metastatic when injected into the tail veins of Fischer rats. When the wild-type ras or the ras mutants, including 116K, were expressed in nontumorigenic E1A-plus-E1B-expressing wtAd5 CREF cells, transformed cells induced tumors in both types of animals. The CREF cells doubly transformed with 116K + wtAd5, unlike transformants containing the wild-type ras and the other ras mutants that still retained GTP binding, were still unable to induce lung metastases. In addition, 116K + wtAd5-transformed CREF cells also did not display any alterations in morphology distinguishable from wtAd5 CREF cells and were not able to grow in agar with increased efficiency. These results indicate that the loss of GTP-binding ability by this mutant p21 ras protein eliminated the ability of these proteins to induce an oncogenic phenotype in an immortal but normal CREF cell line. However, the mutant ras could cooperate with wtAd5 transforming genes in transformed CREF cells to make these cells progress to an oncogenic (but not metastatic) phenotype.

Low‐level β1 protein kinase C expression in cloned rat embryo fibroblast cells enhances transformation induced by the adenovirus type 5 E1A gene

Expression of the E1A gene of adenovirus type 5 (Ad5) in a cloned rat embryo fibroblast (CREF) cell line results in morphological transformation. The efficiency of E1A-mediated transformation of CREF cells is increased if a wild-type Ad5 E1A gene is cotransfected with a rat beta 1 protein kinase C (beta 1 PKC) gene. A direct demonstration of complementation between a functional-transforming Ad5 E1A gene and beta 1 PK in inducing transformation was demonstrated using Ad5 E1A cold-sensitive mutant (E1Acs) genes. The E1Acs gene enhanced transformation only at the transformation-permissive temperature of 37 degrees C and not at the nonpermissive transforming temperature of 32 degrees C. CREF cells constitutively expressing low levels of beta 1 PKC mRNA were transformed at a higher frequency than parental CREF cells after transfection with an Ad5 E1A gene or infection with wild-type Ad5 or the Ad5 host-range cold-sensitive mutant H5hr1. There was no enhancement of transformation in low-level beta 1 PKC-expressing CREF cells when cultures were grown continuously in the presence of the PKC-inhibitor 1-(5-isoquinolynsulfonyl)-2-methylpiperazine dihydrochloride. Transfected CREF cells expressing low levels of beta 1 PKC mRNA displayed CREF-like morphology and did not form colonies when grown in agar. In contrast, retroviral vector-transformed CREF cells expressing high levels of beta 1 PKC mRNA and beta 1 PKC enzyme activity were morphologically transformed and grew efficiently in agar. These findings indicate that the beta 1 PKC gene, when expressed at low levels, can cooperate with the Ad5 E1A gene in the initiation of viral oncogene-mediated transformation.

Suppression of adenovirus type 5 E1A‐mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE)

Viral transformation and DNA-transfection assays were employed to investigate the differential toxic effect of caffeic acid phenethyl ester (CAPE), an extract of the honeybee hive product propolis, on adenovirus type 5 (Ad5)-transformed cloned rat embryo fibroblast (CREF) cells. CAPE inhibited, in a dose-dependent manner, both de novo and carcinogen-enhanced transformation of CREF cells by H5hr1, the cold-sensitive (cs) host-range mutant of Ad5. CAPE had a selective inhibitory effect on Ad5-induced transformation when a wild-type (wt) Ad5 E1A gene or a cs Ad5 E1A gene (at 37 degrees C, but not at 32 degrees C) was cotransfected into CREF cells with a dominant-acting bacterial hygromycin-resistance gene. A requirement for the expression of Ad5 E1A-encoded mRNAs and transforming proteins and sensitivity to CAPE was demonstrated using CREF cells stably transformed by a cs Ad5 E1A gene and an Ad5 E1A gene under the transcriptional control of a mouse mammary tumor virus promoter. To distinguish between the effects of the two Ad5 E1A-encoded proteins of 289 amino acids (aa) and 243 aa, CREF cells were stably transformed with cDNAs encoding either the 13S or the 12S E1A mRNA. CREF cells expressing the 13S E1A-encoded 289-aa protein were more sensitive to the growth-suppressing effect of CAPE than cells producing only the 12S E1A-encoded 243-aa protein. However, the growth-suppressing and toxic effects of CAPE were greatest in cells expressing both E1A-encoded transforming proteins. Analysis of the effect of CAPE on E1A and beta-actin gene expression in wt and cs E1A and H5hr1-transformed CREF cells indicated that low levels of CAPE, which were growth suppressive, did not selectively suppress E1A expression. These results demonstrated that cellular changes induced in CREF cells by the 13S E1A-encoded 289-aa protein of Ad5, when expressed alone or in combination with the 12S E1A-encoded 243-aa protein, rendered transformed cells sensitive to the growth-suppressing and toxic effects of CAPE.

Dissection of functional domains in the adenovirus 2 early 1b 55k polypeptide by suppressor-linker insertional mutagenesis

To determine whether the viral replication functions of the adenovirus E1 B 55K protein play a role in its ability to transform cloned rat embryo fibroblast cells in culture, we constructed an extensive series of insertion mutations throughout the 55K gene. The mutations were recombined into infectious virus and characterized for their abilities to produce stable 55K protein in HeLa cells, replicate virus in HeLa cells, express late viral proteins efficiently, and transform CREF cells following infection. Mutant 55K transforming activity in primary baby rat kidney cells was also assayed following DNA transfection. The functions required for viral replication are encoded in several patches of the 55K linear sequence, while the CREF transforming functions are sensitive to all of the insertions. An insertion at amino acid 380 created a mutant virus which was reduced in transforming activity, but was not reduced for viral replication. Therefore, a function required for efficient transformation of CREF cells can be separated from functions required for late gene expression and viral replication. Transformation of BRK cells following DNA transfection was reduced by complete disruption of the 55K protein gene, but was not significantly affected by any of the insertions.

Enhancement of adenovirus transformation of cloned rat embryo fibroblast cells by gamma irradiation

We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.

Enhancement of viral and DNA mediated transformation of cloned rat embryo fibroblast cells by 3‐aminobenzamide

We have analyzed the effect of the poly(ADP-ribose) synthesis inhibitor 3 aminobenzamide (3AB) on de novo and methyl methanesulfonate (MMS) and gamma irradiation enhancement of viral transformation of a cloned rat embryo fibroblast cell line, CREF, by a cold-sensitive host-range mutant of type 5 adenovirus, H5hr1. Additionally, we have evaluated the effect of 3AB on the transformation of CREF cells following transfection with a gene conferring resistance to hygromycin (hygr) or the neomycin analogue G418 (neor) in combination with a cloned type 5 adenovirus E1A transforming gene (Ad5 E1A) or the Ha-ras (T24) oncogene. 3AB induced a dose- and time-dependent increase in the level of de novo MMS-enhanced and gamma irradiation-enhanced transformation of CREF cells by H5hr1, whereas it did not induce morphological transformation in uninfected control, MMS-pretreated, or gamma irradiation-pretreated CREF cells. Temporal kinetic studies indicated that 3AB was most effective in enhancing de novo and MMS-enhanced and gamma irradiation-enhanced viral transformation when applied early after viral and carcinogen plus viral infection and when present for extended time periods (4-5 wk). 3AB also increased the frequency of resistant colonies following transfection with several cloned genes, including hygr, neor, and protein kinase C (which also contained a neor gene), and the frequency of morphological transformation of CREF cells following cotransfection with a hygr gene and an Ad5 E1A or an activated Ha-ras (T24) gene. In contrast, 3AB exerted the opposite effect, i.e., an inhibitory effect, when applied to NIH 3T3 cells transfected with a hygr or neor gene, alone or in combination with a T24 gene. The ability of 3AB to enhance the frequency of transformation of CREF cells was not associated with a selective effect on the growth of H5hr1-transformed CREF cells in monolayer or agar culture. Similarly, 3AB did not alter the percentage of MMS- or gamma irradiated-pretreated H5hr1-infected cells retaining free Ad5 DNA or the random pattern or quantity of viral DNA integration in control or carcinogen-treated H5hr1-transformed cells. These results suggest that cellular processes regulated by the nuclear enzyme ADPRT, or additional processes modified by 3AB, may be important mediators of stable transformation induced by transfected DNA and both de novo and carcinogen-enhanced viral transformation of specific target cells.

Early events in methyl methanesulfonate enhancement of adenovirus transformation of cloned rat embryo fibroblast cells.

Pretreatment of a cloned rat embryo fibroblast (CREF) cell line with methyl methanesulfonate (MMS) prior to infection with a specific host-range and cold-sensitive type 5 adenovirus mutant (H5hr1), results in a unique carcinogen enhancement of transformation (CET) phenotype (Carcinogenesis 8:967, 1987). By using low-density clonal assays and in situ hybridization techniques with 32P-labeled type 5 adenovirus (Ad5) probes, we demonstrated that 5-10 d following infection the proportion of CREF colonies containing H5hr1 DNA and RNA is increased two- to threefold as a result of pretreatment with MMS. Twenty-five days following infection of CREF cells, Ad5 DNA assays showed that both solvent and MMS-pretreated CREF colonies no longer contained detectable levels of viral DNA or RNA. Analysis of free viral DNA by the Hirt procedure suggested that more free viral DNA persisted in MMS-pretreated H5hr1-infected CREF cells than in solvent-pretreated H5hr1-infected CREF cells. The relative amount of free viral DNA in both types of cultures was directly related to the multiplicity of H5hr1 infection and decreased with time following infection. As observed using in situ hybridization techniques, by 25 d after infection no free viral DNA was detected in either MMS- or solvent-pretreated H5hr1-infected CREF cells. By using a protein synthesis inhibitor (cycloheximide) and an RNA transcription inhibitor (actinomycin D), it was further demonstrated that the ability of MMS to induce a unique CET in CREF cells following infection with H5hr1 was dependent on the synthesis of new protein and RNA. In contrast, inhibition of protein and RNA synthesis did not alter the de novo rate of H5hr1 transformation of CREF cells. Temporal kinetic studies indicated that the ability of MMS to enhance H5hr1 transformation of CREF cells and to increase the percentage of CREF colonies containing Ad5 genetic information is regulated in a strict temporal manner. The results of the present investigation suggest that the ability of MMS to enhance H5hr1 transformation of CREF cells is dependent on the induction of new protein(s) in CREF cells, and enhancement is associated with an increase in the proportion of cells in the infected CREF cell population that initially contain Ad5 DNA/RNA.

38 - Transfer of a Malignant Phenotype by Calcium-Mediated DNA Transfection from Human Prostatic Carcinoma Cells to a Cloned Rat Embryo Fibroblast (CREF) Cell Line

38 - Transfer of a Malignant Phenotype by Calcium-Mediated DNA Transfection from Human Prostatic Carcinoma Cells to a Cloned Rat Embryo Fibroblast (CREF) Cell Line

Unique carcinogen enhancement of transformation phenotype displayed by cloned rat embryo fibroblast (CREF) cells treated with methyl methanesulfonate and infected with a specific host-range mutant of type 5 adenovirus.

Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with wild-type 5 adenovirus (H5wt) or a temperature-sensitive mutant of Ad5 (H5ts125) results in an MMS dose-dependent enhancement of viral transformation. With both viral isolates, MMS enhanced the transformation frequency when normalized for cell toxicity but did not induce a carcinogen dose-dependent increase in the absolute number of foci above solvent-treated controls. In contrast, pretreatment of CREF cells with MMS prior to infection with a host-range mutant of Ad5 (H5hr1) which contains a single basepair deletion in the E1a-transforming region of Ad5 and displays a cold-sensitive transformation phenotype, results in an MMS dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for cell toxicity. To explore the possible mechanism by which H5hr1 displays its unique carcinogen-enhancement of transformation (CET) phenotype we have examined the effect of MMS pretreatment on the frequency of transformation of CREF cells infected with mutants of Ad5 which were engineered to contain either a deletion (H5dl101) or an insertion (H5in106) mutation in the E1a gene region resulting in a cold-sensitive transformation phenotype similar to H5hr1. MMS-pretreated CREF cells infected with H5dl101 or H5in106 did not demonstrate a dose-dependent increase in the absolute number of transformed foci as was observed with carcinogen-pretreated H5hr1-infected CREF cells. These findings suggest that the unique CET phenotype displayed by H5hr1 may result from a second site mutation in a region of H5hr1 other than the E1a-transforming region and/or a novel interaction between gene products resulting from the specific mutation in E1a and other region(s) of the H5hr1 genome. Our investigations also indicate that the CREF/H5hr1 system should prove useful in analyzing chemical-viral interactions in cell transformation.

Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins.

Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G0-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo 32P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G0-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

Mutations in the E1a gene of adenovirus type 5 alter the tumorigenic properties of transformed cloned rat embryo fibroblast cells.

The adenovirus type 5 mutants H5hr1 and H5dl101 contain modifications in the E1a gene affecting the 13S mRNA-encoded 289-amino acid polypeptide and exhibit a cold-sensitive transformation phenotype upon infection of cloned rat embryo fibroblast (CREF) cells. Transformed cell lines expressing solely E1a or E1a and E1b gene products derived from these viruses display enhanced anchorage-independent growth at 37 degrees C versus 32 degrees C and display a cytoskeletal architecture resembling untransformed fibroblastic CREF cells. In contrast, CREF cells transformed by H5wt or the E1a and E1b region of H5wt grow with similar efficiency in agar at 37 degrees C or 32 degrees C and exhibit an epithelioid morphology that is associated with an altered cytoskeleton. Regardless of the expression or presence of other viral early regions, including E1b, E2a, and E4 genes, specific CREF cell lines expressing an altered 289-amino acid protein and a wild-type 12S mRNA-encoded 243-amino acid protein were capable of inducing tumors in nude mice and in immunocompetent syngeneic Fischer rats. In sharp contrast, cells expressing a wild-type 289-amino acid protein were unable to induce tumors in either nude mice or syngeneic rats. The ability to induce tumors did not correlate with alterations in the pattern of viral DNA integration or differential expression of the E1a and E1b genes, nor was the tumor induction a consequence of unique properties of the immortal parental CREF cell line.

Effect on transformation of mutations in the early region 1b-encoded 21- and 55-kilodalton proteins of adenovirus 5.

It is well established that the adenovirus 5 genes responsible for the initiation and maintenance of the transformed cell reside in the early region 1a and 1b genes, but it remains unclear how the polypeptides encoded in these genes mediate their functions. To probe the function of the early region 1b-encoded 55- and 21-kilodalton (kd) polypeptides during this process, a series of viral mutants was engineered so that they contained deletions or insertions at 5.4, 5.7, 7.9, or 9.6 map units. By means of either an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) cleaved with ClaI, or a marker rescue procedure involving H5dl312 (delta 1.2 to 3.8 map units), viral mutants were isolated by their ability to produce plaques on KB cell line 18 cells, which constitutively express only viral early region 1b functions. DNA sequence analysis confirmed that the series of mutants generated differed in their abilities to express the 21- or the 55-kd polypeptides, or both. Upon infection of cloned rat embryo fibroblast cells with viruses containing mutations affecting the 55-kd protein, the transformation frequency decreased as the size of the predicted truncated polypeptide decreased. Although all of the foci generated by the 55-kd protein mutants were indistinguishable from the foci induced by wild-type virus, they displayed an inefficient ability to grow in soft agar, again in relation to the size of the truncated polypeptide. In contrast, if cloned rat embryo fibroblast cells were transfected with viral DNA, the defectiveness in transformation observed after infection with virions was not as dramatic. However, all of the viruses containing 21-kd mutations were transformation defective, regardless of the mode by which the viral nucleic acid was introduced into the cell.

Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products.

Type 5 adenovirus mutants that differentially express E1A 13S, 12S, or 9S mRNAs were constructed to study the role of their gene products in transformation. H5dl520 expresses the 243-amino-acid (AA) protein encoded in the 12S mRNA but not the 13S mRNA-encoded 289-AA protein. This mutant transformed both cloned rat embryo fibroblast (CREF) cells and baby rat kidney (BRK) cells at a frequency 40-100 times greater than did wild-type viruses. In addition, all of the foci produced were fibroblastic and grew very slowly at 32 degrees C. In contrast, H5dl21, which was mutated so that only the 54-AA protein encoded by the 9S mRNA was synthesized, did not transform either cell type. DNA transfection studies with plasmids from which these mutants were constructed demonstrated that the differences in transformation frequencies were not as marked. The plasmid pD1/D2, which directs the synthesis of the 54-AA protein only, was found to transform baby rat kidney cells at low frequency, provided the gene was linked to a fragment from the simian virus 40 genome containing the transcriptional enhancer element.