Production of metastatic phenotype with DNA from metastatic cells but not with oncogenic DNA

Although the tumorigenic phenotype can be transferred by transfecting DNA from a variety of human tumours into 3T3 mouse fibroblasts, the ability to transfer the metastatic phenotype in a similar manner in syngeneic animals is largely unknown. We now report that transfection of a rat mammary epithelial cell line Rama 37, which produces nonmetastasizing adenomatous tumours in syngeneic rats, with DNA from a metastasizing rat mammary cell line, Rama 800, yields some transfectants with reproducible metastatic capabilities. Nonspecific DNA or the oncogenes EJ-ras-1 or Polyoma Large T Antigen fail in this respect, although their transfectants often possess increased tumorigenic potential.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts.

We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met.     

Three different human tumor cell lines contain different oncogenes

We have obtained foci of transformed mouse cells after transfection of human DNA from colon and bladder carcinoma cell lines and a promyelocytic leukemia cell line. These foci can be shown to contain a large number of human DNA sequences by use of highly repetitive human DNA sequence probes. Cell DNA from primary foci can be used in a subsequent cycle of transfection resulting in secondary foci that contain relatively little human DNA. Secondary foci appear to contain only the human sequences proximal to those responsible for the transformed phenotype. A set of characteristic DNA restriction fragments is found in common among secondary foci derived from each tumor cell line DNA. Comparison of the common DNA fragments found in secondary foci derived from three different human tumor cell lines indicates that these three cell lines contain three different transforming genes.     

Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies.

Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.     

Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA

We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene.     

高转移人肺癌细胞系DNA转移特性的研究

 用高转移肺癌细胞DNA转染NIH/3T3鼠细胞,获得的转化细胞在裸鼠体内表现不同的成瘤潜伏期,鉴定了整合到鼠细胞中入DNA序列,并得到一株在裸鼠体内有转移活性的转化细胞株。     

Isolation of a transforming sequence from a human bladder carcinoma cell line

We have isolated the component of human bladder carcinoma cell DNA that is able to transform mouse fibroblasts. The oncogenic sequence was isolated initially from a lambda phage genomic library made from DNA of a transfected mouse cell carrying the human oncogene. A subcloned insert of 6.6 kb that carried transforming activity was amplified in the plasmid vector pBR322. The subcloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has failed so far to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human tumor cell lines derived from colonic and lung carcinoma and from neuroblastoma. In contrast, the EJ bladder oncogene appears closely related to one that is active in the human T24 bladder carcinoma cell line. The oncogene appears to have undergone little, if any, amplification in several bladder carcinoma cell lines.     

Transfer of active oncogenes and promoters into the mouse cell genome

Different cell DNA's (normal NIH 3T3 DNA; human osteosarcoma cell DNA; human malignant glioma cell DNA with amplified c-Ha-ras) were cotransfected onto NIH 3T3 cells with cloned long terminal repeat (LTR) sequences of Rous sarcoma virus. LTR RSV and normal NIH 3T3 DNA c-fos oncogen expression was detected in tumors induced in nude mice. In the same system human tumour cell DNA with amplified c-Ha-ras gene was used, that to the integration and amplification of LTP sequences with simultaneous maintenance of c-Ha-ras amplification. Nude mouse tumour DNA with integrated LTR sequences was active in successive rounds of transfection.     

Late transient expression of human hepatitis B virus genes in monkey cells

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.     

Recombinant mussel adhesive protein as a gene delivery material

Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant mussel adhesive protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine 2000. Our results indicate that this mussel adhesive protein may be used as a potential protein-based gene-transfer mediator.     

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.     

DNA repair and repair fidelity in metastatic variants of the B16 murine melanoma

We have examined the concept of genomic instability in relation to the metastatic progression of low (F1) and high metastasis (BL6, ML8) clones of the B16 mouse melanoma, by using a mutation assay, and DNA strand break repair and repair fidelity assays. The frequency of induced ouabain resistant colonies between the variant cell lines was consistent with the difference between their metastatic properties. Survival data for X-irradiation and bleomycin were similar among the 3 cell lines. When X-rays or bleomycin were used to induce strand breakage, no difference was detectable in either the rate or extent of DNA repair using the techniques of alkaline unwinding and alkaline elution for total strand breaks, and neutral elution for double strand breaks. DNA repair fidelity was measured using the PMH16 plasmid. A Kpn I restriction site was used to introduce a break within the gpt gene of the plasmid, prior to transfection. We found that ～ 100% and ～ 65% of the highly metastatic ML8 and BL6 clones, respectively, religated the gene with the required fidelity, compared with only ～ 25% of the low metastasis F1 clones. In summary, the metastatic variants show similar sensitivities to X-irradiation and bleomycin, but a differential response to EMS. This difference is not reflected in any subsequent DNA strand break religation, but the variants do differ in their fidelity of repair. However, although the fidelity of DNA religation is related to metastatic potential, it is not consistent with the mutation frequency data. © 1993 Wiley-Liss, Inc.     

SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.     

Control of metastasis by Asn-linked, beta1-6 branched oligosaccharides in mouse mammary cancer cells

Studies in cell lines and malignant human tissues have shown that increased cell-surface Asn-linked beta1-6(GlcNAcbeta1-6Man) branching is associated with increased tumorigenic and metastatic properties. In this study, three mouse mammary cancer cell lines were transfected with an expression vector containing the mouse cDNA for N-acetylglucosaminyltransferase V (GlcNAcT-V EC 2.4.1.155), the glycosyltransferase responsible for initiating beta1-6 branching on Asn-linked carbohydrates. The cell lines were screened for increased cytotoxicity to L-PHA, a lectin specific for beta1-6 branching structures. Cell lines exhibiting increased L-PHA cytotoxicity expressed increased levels of beta1-6 branching structures. Northern blots detected the presence of GlcNAcT-V transcribed from the expression vector in the L-PHA sensitive cell lines. After injection into the tail veins of mice, transfected cell lines with increased beta1-6 branching on the cell surface formed elevated levels of lung tumors relative to control transfected cell lines (P < 0.002). Western blots of membrane proteins from GlcNAcT-V transfected and control cells probed with the lectins DSA and WGA did not show an increase in polyN-acetyllactosamine and sialic acid content in the transfected cell lines. These results demonstrate that a specific increase in beta1-6 branching due to an elevation in GlcNAcT-V expression increases metastatic potential.     

Integration of Rous sarcoma virus DNA during transfection

We have investigated the organization and integration sites of Rous sarcoma virus (RSV) DNA in NIH 3T3 mouse cells transformed by transfection with unintegrated and integrated donor RSV DNAs. RSV DNAs of different cell lines transformed by unintegrated donor DNA were flanked by different cellular DNA sequences, indicating that RSV DNA integrates at multiple sites during transfection. The RSV genomes of cells transformed by transfection were colinear with unintegrated RSV DNA, except that deletions within the terminal repeat units of RSV DNA were detected in some cell lines. These results suggested that the terminal repeat sequences of RSV DNA did not necessarily provide a specific integration site for viral DNA during transfection. In addition, cell lines transformed by integrated RSV DNAs contained both the RSV genomes and flanking cellular sequences of the parental cell lines, indicating that integration of integrated viral DNA during transfection occurred by recombinational events within flanking cellular DNA sequences rather than at the terminal of viral DNA. Integration of RSV DNA during transfection thus appears to differ from integration of RSV DNA in virus-infected cells, where the terminal repeat units of viral DNA provide a highly specific integration site. Integration of donor DNA during transfection of NIH 3T3 cells instead appears to proceed by a pathway which is nonspecific for both donor and recipient DNA sequences.     

Cloning and characterization of human syndecan-3.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.     

Nucleofection-based gene targeting in human pre-B cells

Electroporation is a powerful and convenient means for transfection of nonviral vectors into mammalian cells, providing an essential tool for numerous applications including gene targeting via homologous recombination. Recent evidence clearly suggests that high-efficiency gene transfer can be achieved in most cell lines by nucleofection, an electroporation-based transfection method that allows transfected vectors to directly enter the nucleus. In this paper, we analyze the effectiveness of nucleofection for gene targeting using human pre-B cells. For this, we tested 93 different transfection conditions, and found several conditions that gave high (~ 80%) transfection efficiency with low cytotoxicity (~ 70% survival rate). Remarkably, under the optimal nucleofection conditions, the gene-targeting efficiency was ~ 2-5-fold higher than that achieved with conventional electroporation methods. We also found that nucleofection conditions with high transfection efficiency and low cytotoxicity tend to provide high gene-targeting efficiency. Our results provide significant implications for gene targeting, and suggest that nucleofection-based nonviral gene transfer is useful for systematic generation of human gene-knockout cell lines.     

Transformation and implication of the K-fgf proto-oncogene in NIH-3T3 cells,and induction of metastatic potential

A plasmid containing the K-fgf proto-oncogene linked to the dihydrofolate reductase gene has been constructed, and used in transfection experiments to investigate the effects of K-fgf expression on the tumorigenic and metastatic properties of NIH-3T3 fibroblasts. Analysis of cells transfected with K-fgf revealed that expression of the K-fgf proto-oncogene can, in a single step, induce both tumorigenic and metastatic characteristics, as determined in soft agar cloning experiments, and in tumorigenicity and experimental lung metastasis assays with BALB/c nu/nu mice. Selection for resistance to increasing concentrations of methotrexate lead to the isolation of a series of cell lines containing amplifications of both the dihydrofolate reductase gene and the linked K-fgf gene, which synthesized elevated levels of growth factor message and protein. The most highly resistant and gene amplified cell lines exhibited lower than expected levels of K-fgf mRNA, and also appeared to have down-regulated cell surface growth factor receptors. Further support for the concept that altered K-fgf expression can induce fully malignant and metastatic cells was obtained in experimental metastasis assays, where K-fgf transfected and gene amplified cell lines were highly aggressive.