Expression of growth-regulated genes in normal and SV40 transformed hamster fibroblasts.

Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth-regulated genes in SV40-transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1-specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk-ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk-ts13/1%), was also included in these studies. The low serum adapted SV40-transformed cells and the spontaneous tk-ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental.(ABSTRACT TRUNCATED AT 250 WORDS)     

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells.

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk-ts13) which is temperature sensitive for growth. These tk-ts13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the G0 state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice     

Enhancer-controlled expression of the simian virus 40 T-antigen in the green alga Acetabularia.

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T-antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T-antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

Effects of sodium n-butyrate on entry into S phase in resting rat 3Y1 cells infected with simian virus 40.

In quiescent rat 3Y1 fibroblasts infected with simian virus 40 (SV40), sodium butyrate elongated the time lag before entry into S phase in a concentration-dependent fashion. In spite of the elongated time lags, SV40-infected cells entered S phase in a very synchronous mode, irrespective of the butyrate concentrations. The elongated time lag seemed to be at least partially due to a delayed synthesis and a delayed accumulation of large T antigen caused by butyrate. The entry into S phase was also delayed even when butyrate was added to the cultures after expression of T antigen to an extent sufficient for untreated cells to enter S phase. This suggests that butyrate may also inhibit a cellular event(s) that is required for entry into S phase after expression of the T antigen. In contrast, serum-stimulated cells were more sensitive to butyrate with respect to entry into S phase than SV40-infected cells, and the distribution of the time lag among cell populations increased (i.e., asynchrony in entry into S phase increased) with an increase in the butyrate concentration.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

Physiological concentrations of butyrate favorably modulate genes of oxidative and metabolic stress in primary human colon cells

Butyrate, a metabolite of gut flora-mediated fermentation of dietary fibre, was analysed for effects on expression of genes related to oxidative stress in primary human colon cells. An induction of detoxifying, antioxidative genes is expected to contribute to dietary chemoprevention. Cells were treated with butyrate (3.125-50 mM; 0.5-8 h), and kinetics of uptake and survival were measured. Gene expression was determined with a pathway-specific cDNA array after treating colon epithelium stripes with nontoxic doses of butyrate (10 mM, 12 h). Changes of hCOX-2, hSOD2 and hCAT expression were confirmed with real-time polymerase chain reaction (PCR) and by measuring catalase-enzyme activity. Primary colon cells consumed 1.5 and 0.5 mM butyrate after 4- and 12-h treatment, respectively. Cell viability was not changed by butyrate during 0.5-2-h treatment, whereas cell yields decreased after 1 h. Metabolic activity of remaining cells was either increased (4 h, 50 mM) or retained at 97% (8 h, 50 mM). Expression of hCAT was enhanced, whereas hCOX-2 and hSOD2 were lowered according to both array and real-time PCR analysis. An enhanced catalase-enzyme activity was detected after 2 h butyrate treatment. Healthy nontransformed colon cells well tolerated butyrate (50 mM, 2 h), and lower concentrations (10 mM, 12 h) modulated cyclooxygenase 2 (COX-2) and catalase genes. This points to a dual role of chemoprotection, since less COX-2 could reduce inflammatory processes, whereas more catalase improves detoxification of hydrogen peroxide (H(2)O(2)), a compound of oxidative stress. Changes of this type could reduce damaging effects by oxidants and protect cells from initiation.