Adenovirus type 12 gene 401 function and maintenance of transformation.

Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants.

The growth properties of hamster cells transformed by wild-type Simian virus 40 (SV40), by early SV40 temperature-sensitive mutants of the A complementation group, and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, whereas that rescued from revertant and wild-type transformants was not thermolabile in this regard. T antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 degreeC, was temperature sensitive by in vitro immunoassay, whereas that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44 degrees C, whereas T antigen from tsA30 mutant-transformed cells was relatively thermolabile. These results further indicate that T antigen is a product of the SV40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown. This is consistent with the suggestion that the amount of T antigen synthesized in CHLA30L1 is large enoughto allow partial expression of the transformed phenotype at the restrictive temperature. Alternatively, the increase in T antigen concentration may be secondary to one or more genetic alterations that independently affect the transformed phenotype of these cells.     

Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus.

A fibroblast line of the 3T3 type with a low saturation density was established from Fisher rat embryo cells. After infection with either wild-type or tsa mutant polyoma virus, transformants were isolated and cloned at 33 degrees C on the basis of their ability either to grow as dense foci on plastic in liquid medium (type N) or to form colonies in soft agar (type A). Polyoma T antigen was detected in all of the transformed lines. The following growth characteristics were studied for both types at 33 and 41 degrees C: saturation density, growth in soft agar and at a low serum concentration, colony-forming ability, and generation time. tsa-N transformants behaved at 33 degrees C similarly to transformed cells, but reverted at 41 degrees C to the nontransformed phenotype for all of these characters. tsa-A transformants and all of the wild-type transformants exhibited the transformed phenotype at both low and high temperatures. These results led us to distinguish at least two types of virus-induced transformants. In one of them, the activity of the protein affected by the tsa mutation appears to be necessary for the expression of several of the characters defining the transformed state.     

Simian virus 40 gene A function and maintenance of transformation.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.     

Delayed Leaf Senescence in Tobacco Plants Transformed with tmr,a Gene for Cytokinin Production in Agrobacterium

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.     

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

The role of phytohormones in tumor formation in radish

Tumor formation was studied in inbred radish lines that produce tumors on plant roots during flowering. In all radish lines under consideration, the sequences homologous to oncogenes tmr/tml of Agrobacterium tumefaciens were revealed by Southern hybridization. No sequences homologous to the tms locus of A. tumefaciens and the oncogenes of A. rhizogenes were determined. It was found that auxin sensitivity and the tumor-producing capacity were coinherited. We suggest that tumor phenotype arise as a result of a combination between agrobacterial "cytokinin" oncogenes and certain alleles of "auxin" radish genes.     

Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment

Constitutive expression of basic fibroblast growth factor (bFGF), a common characteristic of metastatic melanomas, was reproduced in vitro by infection of normal murine melanocytes with a recombinant retrovirus carrying a cDNA for bFGF. Expression of bFGF in these cells conferred autonomous growth in culture and extinguished differentiated functions, such as the synthesis of melanin and formation of dendrites. Independence from exogenous bFGF and loss of differentiated functions in vitro were induced also by transformation of melanocytes with the oncogenes myc, Ela, ras, and neu, although bFGF was not expressed by the respective transformants. As shown in skin reconstitution experiments onto syngeneic mice and subcutaneous injections into nude mice, the various transformants differed in their behavior in vivo. The bFGF transformants did not form tumors. They reverted to having a normal, melanotic phenotype and restricted growth. Myc and Ela transformants grew as tumors in nude mice but not in syngeneic, immunocompetent animals. Ras-transformed melanocytes were always tumorigenic, whereas the formation of tumors by neu transformants was suppressed by the concomitant grafting of keratinocytes in reconstituted skin of syngeneic mice. These data show that melanocytes genetically manipulated to produce bFGF acquire properties in vitro similar to those of metastatic melanoma cells or those induced by various oncogenes but that constitutive production of bFGF by itself is insufficient to make melanocytes tumorigenic. The experiments also show that melanocytes transformed by the selected oncogenes respond differentially to various environments in vivo.     

Genetic analysis of T-DNA transcripts in nopaline crown galls

Plant crown gall tumor cells result from the insertion and expression of a defined DNA sequence, called T-DNA, which is derived from the Ti plasmid, harbored by Agrobacterium tumefaciens strains. To study the function of the genes of the T-DNA of the nopaline Ti plasmid, pTiC58, a collection of mutants was isolated so that T-DNA genes are inactivated either separately or in various combinations. It was found that no single T-DNA gene or T-region border is absolutely essential for stable tumor formation. We have identified the gene responsible for synthesis in transformed cells of the phosphorylated sugar, agrocinopine, and at least three additional genes controlling the morphology of plant tumors. Two of these latter genes work together to inhibit shoot formation and ensure efficient tumorous growth. Inactivation of these genes can be suppressed by the addition of auxins. The third gene inhibits root formation and appears to play a role in the cytokinin-independent growth of transformed cells. Mutants missing all three genes do not induce tumors, nor shoot or root formation, although the mutant T-DNA sequence is transferred to plant cells.     

Transformation phenotype of polyoma virus-transformed rat fibroblasts: plasminogen activator production is modulated by the growth state of the cells and regulated by the expression of an early viral gene function

The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants independently isolated under various growth conditions led to the conclusion that there is no correlation between the regulation processes involved in plasminogen activator production and ability to grow without anchorage. The results obtained also suggested that the production of plasminogen activator is under the control of a functional large T antigen.     

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed byAgrobacterium tumefaciens

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the “rooty” locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed by the mutant bacteria showed that the auxin independent phenotype is suppressed, but can be reinitiated in culture by exposure to an exogenous supply of auxin. In addition the developmental state of the cells from such regenerated plants can exert a profound influence on their cytokinin autonomy phenotype.     

Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells

During the development of tobacco plants, cells undergo epigenetic changes that alter their requirement in culture for the cell-division factor cytokinin. Cultured leaf cells alternate between cytokinin-requiring (C-) and cytokinin-independent (C+) states at extremely high rates of approximately 10-2 per cell generation by a process called pseudodirected variation. Here we show that plants regenerated from most C+ clones express the Habituated leaf (Hl) trait, i.e., leaf tissues exhibit the C+ phenotype rather than the wild-type C- phenotype in culture. This new trait then segregates as a monogenic dominant trait indicating that conversion of C- cells to C+ cells is associated with a meiotically transmissible, genetic modification. Two independent mutants, Hl-2 and Hl-3, derived from C+ variants arising in culture were unstable in planta and reverted gametically at rates roughly comparable to pseudodirected variation in culture. Cells of the Hl-2 mutant, but not of a stable Hl-1 mutant, reverted phenotypically at high rates in culture. This revertant C- phenotype persisted in some plants regenerated from cloned revertant lines, and then showed irregular segregation in two successive sexual generations. These results show for the first time that meiotically transmissible epimutations can occur reversibly and at high rates in culture.     

Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.     

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

DNA fragments (0.5-4.5 kb) of normal human lymphocytes induced pre-neoplastic mouse NIH/3T3 cells after transfection to grow in soft agar medium at low efficiency (0.0007 colonies/micrograms DNA/10(6) cells). In secondary transfections high mol. wt. DNA (greater than 20 kb) of cells transformed by DNA fragments induced neoplastic transformation with high efficiency (0.16-1.1 soft agar colonies/micrograms DNA/10(6) cells). These results confirm previous data obtained by others with chicken and mouse donor DNA. We describe here that independent secondary transformants harbored human Alu repetitive DNA sequences on similar restriction fragments and formed progressively growing tumors in BALB/c mice or nude mice. The corresponding primary transformants were not tumorigenic, however, and the ability to proliferate in semi-solid agar medium was gradually lost when the cells were grown as non-confluent monolayers. Furthermore, in contrast to secondary transformants, DNA from primary transformants showed only relatively weak hybridization to a human Alu repetitive DNA probe. We conclude that in primary transformants the transformed phenotype is expressed in an unstable fashion whereas secondary transformants appear to be stably transformed.     

Transformation of kiwifruit using the <Emphasis Type="Italic">ipt</Emphasis> gene alters tree architecture

To manipulate the architecture of woody plants by controlling endogenous cytokinin levels, the isopentenyl transferase gene (ipt) from Agrobacterim tumefaciens was introduced to kiwifruit using stable transformation. Consequently, eight transgenic lines were obtained. Transgenic shoots harboring the ipt gene were recalcitrant to rooting under tissue-culture conditions; thus, their in vitro-cultivated shoots were directly grafted onto potted wild-type kiwifruit seedlings to evaluate their morphological features, and three lines (tmr2-4, tmr2-G, tmr3-C) were successfully grafted. The grafted transgenic plants had dwarfing and branching phenotypes, both of which are typical features of cytokinin overproduction. In addition, the number of buds increased and internode length was shorter in the grafted transgenic plants. The content of a precursor, trans-zeatin riboside, and an active cytokinin, trans-zeatin, increased in one transgenic line, in which the level of ipt gene expression was high, indicating that morphological changes were related to expression levels of the ipt gene and cytokinin content. Possibilities for potential utilization of the ipt gene in manipulating tree shape are discussed.     

Retroviral protein P15E and tumorigenesis. Expression is neither required nor sufficient for tumor development

Murine tumor cells frequently express retroviral protein p15E, a protein with antiinflammatory activity. This has led to the hypothesis that p15E expression allows nascent tumor cells to escape host immunologic defenses. To evaluate the role of p15E expression in tumorigenesis, NIH3T3 cells transformed by various oncogenes and BALB/c lines transformed by carcinogens or SV40 were examined for p15E expression and tumorigenicity. All of the NIH3T3 transformants and most of the BALB/c transformants did not express p15E, indicating that transformation per se does not inevitably induce the expression of p15E. Although not expressing p15E, some of these transformants were capable of forming tumors in immune competent hosts, indicating that p15E is not universally required for tumor growth. Four of the transformed cell lines negative for p15E expression and deficient in tumor-forming capacity were transfected with a gene coding for Moloney retroviral p15E. Despite the expression of p15E, there was no augmentation of their tumorigenic capacity, showing that p15E is not sufficient to ensure tumor formation by a transformed cell. These results argue against a general role for retroviral p15E expression in tumorigenesis.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.