Primary Rat Embryo Fibroblasts Research Articles

Inhibition of ras-induced proliferation and cellular transformation by p16INK4.

The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocked entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block was relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppressed cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth.

Oncogenic capacity of the E2F1 gene.

Previous experiments have identified the E2F transcription factor as a potential downstream target for the action of cellular regulatory activities, such as the Rb tumor suppressor protein, that control cell growth and that, when altered, contribute to the development of human tumors. In light of these findings, we have assayed the ability of the E2F1 and DP1 genes, which encode heterodimeric partners that together create E2F activity, to act in an oncogenic fashion. We find that E2F1, particularly in combination with the DP1 product, cooperates with an activated ras oncogene to induce the formation of morphologically transformed foci in primary rat embryo fibroblast cultures. In addition, an E2F1 chimeric protein, in which sequences involved in Rb binding have been replaced with the herpesvirus VP16 activation domain, exhibits increased transformation activity. Cells transfected with E2F1 and DP1 or the E2F1-VP16 chimera form colonies in soft agar and induce tumor formation in nude mice. We conclude that deregulated E2F1 expression and function can have oncogenic consequences.

Recovery of a rare clone from a population of unstable retroviral vector-expressing mammalian cells using a new RNA extraction and slot-blot protocol

Although a useful and important method of gene transfer, retroviral vectors can be genetically unstable. In the course of experiments using DOEJS, a retroviral vector able to confer expression of a H- ras oncogene and a neomycin resistance gene ( neo) on mammalian cells (Compere et al., 1989), it was found that the vast majority of infected rat embryo fibroblasts, recovered on the basis of neo activity (i.e., G418 resistance), did not express ras mRNA. It was subsequently observed that most cells in the ψ2 cell line used to propagate DOEJS failed to produce virus capable of expressing both ras and neo in primary rat embryo fibroblasts. A simplified RNA extraction and slot-blot technique was developed to screen mRNA from several hundred fibroblast clones and, in doing so, infected fibroblast clones producing both neo and ras mRNA were identified at low frequency. The DOEJS/ψ2 packaging line was subsequently subcloned and individual clones screened for their ability to confer appropriate gene expression on target cells. Subclone DOEJS/ψ2-B6 was eventually isolated after screening 24 DOEJS subclones and 240 infected rat embryo fibroblast colonies. DOEJS/ψ2-B6 was shown to induce reliably phenotypic transformation, G418 resistance, and ras and neo mRNA expression in primary rat embryo fibroblasts. The RNA extraction and screening procedure was thus useful for recovering an infrequent subclone producing a retrovirus with the original properties.

SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts.

The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Jun-encoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth.

Modulation of cyclin gene expression by adenovirus E1A in a cell line with E1A-dependent conditional proliferation.

To investigate how adenovirus E1A controls cell proliferation, we have fused E1A to the hormone-binding domain of the human estrogen receptor (ER) and introduced the E1A-ER chimeric gene together with an activated ras gene into primary rat embryo fibroblasts. Cell lines derived from this transfection proliferate in an estrogen-dependent manner. Estrogen-dependent activation of E1A-ER led to a rapid induction of both cyclin E and cyclin A gene expression. In contrast, levels of cyclin D1 were strongly reduced by activation of E1A-ER. Similar changes in cyclin gene expression were observed when primary human fibroblasts were infected with wild-type adenovirus and when adenovirus E1A was stably expressed in NIH 3T3 cells. Our findings suggest that activation of cyclin A and E, but not D1, gene expression by E1A precedes and may be responsible for E1A-dependent cell proliferation. In contrast, we found that quantitative disruption of complexes between the E2F transcription factor and the retinoblastoma protein is not required for E1A-dependent S-phase entry.

Cyclin Expression and G 2 -Phase Delay after Irradiation

Many studies have demonstrated the effect of oncogene transfection on the radiation sensitivity of primary rat embryo fibroblasts. Our results indicate that transformation by H-ras plus v-myc oncogenes confers radiation resistance to a much greater extent than transformation by either gene alone. We have further shown that the radioresistant phenotype is accompanied by a prolonged G2-phase delay. This is consistent with the hypothesis that the extent of this delay is an important determinant of radiation sensitivity. The study of cyclin expression during the progression of cells through G2 and M phase after irradiation has also revealed several possible mechanisms for induction of G2-phase arrest. Control of cyclin B levels was seen both at the mRNA level as evidenced by decreased cyclin B mRNA expression after irradiation in the S phase, and at the protein level as demonstrated after irradiation in G2 phase. It remains to be determined how these mechanisms might be differentially regulated in radioresistant and sensitive cells.

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties.

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.

Human papillomavirus type 18 E7 protein requires intact Cys-X-X-Cys motifs for zinc binding, dimerization, and transformation but not for Rb binding

Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.

Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site.

We cloned the 5' nontranslated region (NTR) from the genome of swine vesicular disease virus (SVDV), a member of the family Picornaviridae, and used it to construct a bicistronic retroviral vector. The vector is characterized by coexpression of two genes from a single transcript. We found that inclusion of the 5' NTR of SVDV did not negate the viral vector titer. Protein analysis indicated that the 5' NTR could efficiently direct internal initiation, thus allowing the downstream gene to be translated. Translation of the internally initiated porcine growth hormone gene was about 30-fold less than that when the porcine growth hormone gene was at the upstream position in NIH 3T3 cells but was about equivalent to that in HeLa cells, implying that some cellular factors that stimulated internal initiation of the SVDV 5' NTR are present in HeLa cells. However, in G418-selected clones, the Neor-encoding gene was expressed with equivalent efficiency either at a downstream position or at an upstream position in either NIH 3T3 or HeLa cells. Compared with the conventional double-gene vector or the U3-based vector, the bicistronic vector coexpressed two genes much more efficiently, owing to elimination of promoter interference. Furthermore, this type of vector infected and expressed the target genes efficiently in two primary cell lines, rat embryo and human skin fibroblast cells, which we tested. These experimental data suggest a better design for the retroviral vector and provide evidence that internal initiation of the SVDV 5' NTR was stimulated cell specifically.

ヒトパピローマウイルスと癌

More than twenty genotypes of human papillomaviruses (HPVs) are associated with mucosal lesions and more than thirty genotypes with cutaneous lesions. We established a method using consensus polymerase chain reaction (PCR) with restriction endonuclease analysis of PCR products, which can detect and distinguish mucosal high risk HPV types 16, 18, 31, 33, 52b and 58 (J. Gen. Virol. 72, 1039, 1991). By using this method, we analyzed 39 cases of cervical carcinomas and 27 cases of cervical intraepithelial neoplasias (CIN). It was found that the HPV16 was most prevalent in malignant (19 cases) and premalignant (19 cases) cervical lesions, but other types including HPV18, 31, 33, 52b and 58 were also detected especially in cervical carcinomas. We also established a highly sensitive method for specific detection of HPV types 16, 18, and 33 (Jpn. J. Cancer Res., 81, 1, 1990) . The method detected HPV16 DNA in only 5 out 92 cases of cytologically or histologically normal cervices (Jpn. J. Cancer Res., 82, 532, 1991) . HPV16 was also predominantly detected in penile (68 out of 111 cases) and tongue (8 out of 24 cases) squamous cell carcinomas.HPV16 E6E7 region produces at least three different species of E6E7 mRNAs (unspliced E6-E7, spliced E6* I and E6* II) in the HPV16-containing carcinoma cells. We synthesized each of the E6E7 cDNAs and studied on their biological activities. The E6*I cDNA showed highest activities of ras-collaborative transformation of primary rat fibroblasts and induction of cellular DNA synthesis. We analyzed HPV16 E6E7 mRNAs in three cervical carcinomas and six CINs which contained HPV16 DNA by PCR with reverse transcription (RT-PCR) . The major transcript from HPV16 E6E7 region was E6* I except for one CIN tissue where only the full-length E6-E7 mRNA was detected.Different from E7 genes of mucosal high-risk HPVs, E7 genes of HPV5 and HPV8, which are associated with epidermodysplasia verruciformis (EV) were inactive for transforming ability in cell lines. We cloned HPV5 and 8 E7s separately into the expression vector pcD2Y or pGEM3Zf and examined their biological and biochemical activities. Although HPV5 and 8 E7s failed to immortalize primary baby rat kidney cells, they collaborated with activated ras gene to transform primary rat embryo fibroblasts (Jpn. J. Cancer Res., 82, 1340, 1991) and baby rat kidney cells. In vitro binding experiment of retinoblastoma tumor suppressor protein (RB) detected the complex formation of HPV5 and 8 E7s with RB but with reduced affinities compared to HPV16 E7.

The mdm-2 Oncogene Can Overcome Wild-Type p53 Suppression of Transformed Cell Growth

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.

The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth.

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.

C- ets-1 protooncogene expression alters the growth properties of immortalized rat fibroblasts

Ets family genes have been cloned and characterized from a variety of species ranging from human to Drosophila. The ets proteins encode transcription factors that activate transcription via specific binding to GGAA core sequence present in various promoter/enhancers. To investigate the role of ets protooncogene expression on the growth properties of rat embryo fibroblasts (REF), we constructed and introduced ets expression vectors into primary, as well as immortalized REF cells. The transfected cells contained multiple copies of the vector DNA, and the Northern blot analysis demonstrated overexpression of the c-ets-1-specific mRNA. Although the expression of the ets genes was unable to immortalize primary rat embryo fibroblasts, the expression of ets-1 in REF-1 cells enabled their growth in serum-free medium and effected tumorigenic activity in nude mice.

A mutant p53 protein is required for maintenance of the transformed phenotype in cells transformed with p53 plus ras cDNAs.

Mutant p53 and activated ras cDNA clones cooperate to fully transform primary rat embryo fibroblasts in cell culture, whereas neither cDNA alone results in the full transformation of these cells. The mutant p53 protein may be required to initiate the transformation event with ras. Alternatively, mutant p53 gene expression may be required to maintain the properties of the transformed phenotype. To distinguish between these possibilities, primary rat embryo fibroblasts were transformed with mutant p53 plus ras cDNAs, where the expression of the p53 gene was regulated by an isopropyl beta-D-thiogalactoside-responsive promoter. When expression of the mutant p53 cDNA was inhibited and no detectable exogenous p53 protein was produced, both the growth rate and the morphology of the cells reverted to a normal phenotype. These results demonstrate that a mutant p53 protein is required for the maintenance of the transformed phenotype in cells transformed with p53 plus ras cDNAs.

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha- ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60 c-src protein as cells transformed by the same p53 and cotransfected activated Ha- ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60 c-src -specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha- ras oncogene in cell transformation.

P53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70. p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.

Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization

To clarify the relationship between the various activities of the polyomavirus large T antigen and the contribution of this oncogene to neoplastic transformation, we constructed a series of mutants with small deletions or single-amino-acid substitutions in two separate regions of the protein. These sequences were targeted because they showed considerable similarity to conserved regions 1 and 2 of adenovirus E1A which are thought to be binding sites for the retinoblastoma gene product (pRB). The pRB-binding properties of the large T mutants were assessed with an in vitro coimmunoprecipitation assay. pRB binding was readily detected with wild-type large T, but coprecipitation was completely abolished by as little as a single amino acid substitution (Asp-141----Glu or Glu-146----Asp) in region 2 of the polyomavirus large T antigen. Mutants defective in pRB binding were unable to immortalize primary rat embryo fibroblasts, suggesting that association with pRB is an important component of immortalization mediated by polyomavirus large T. The mutations in region 1 affected pRB binding only marginally, yet some of them severely impaired immortalization, indicating that pRB binding may be essential but not sufficient for immortalization.