Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T-antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.     

Effect of the pSV2-neo Plasmid on NIH 3T3 Cell Motion Detected Electrically

Advantage was taken of DNA transfection techniques to investigate the effect of the pSV2-neo plasmid and its derivatives on recipient NIH 3T3 cell motion. Cell spreading and motion were followed by a newly developed electrical method to monitor cell morphology, referred to as electric cell-substrate impedance sensing. Using this method, we found that the eukaryotic--prokaryotic shuttle vector pSV2-neo had a strong effect on the recipient NIH 3T3 cell spreading and cell motion. However, two new neo plasmids, pSK-neo and pSP-neo, which were constructed by modifying the pSV2-neo plasmid, did not have a significant effect on the recipient cell activities. The results suggest that there may be some sequences in pSV2-neo which affect recipient cell behavior.     

Effect of transfection manipulations on mouse cell cycle progression.

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphatedimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak S phase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-3H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1-S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.     

Development of optimized transfectoma cell lines for production of chimeric antibodies in hollow fiber cell culture systems

Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt)and the neomycin (neo) selection marker genes. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3'-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. (c) 1995 John Wiley & Sons, Inc.     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Large scale transfection of mouse L-cells by electropermeabilization

Mouse L-cells were transfected by electropermeabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). The DNA concentration used was 1 microgram/ml, the field strength was 10 kV/cm, the duration of the pulse was 5 microseconds. Transfection yield was optimal at a temperature of 4 degrees C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seconds) or longer (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 degrees C. The yield of transfectants in response to 10 pulses was up to 1000 per 10(6) cells (using 3.3 pg DNA per cell). The influence of the growth phase of the cells on the transfection yield and/or the subpopulation of the mouse L-cell line used was shown. Furthermore the clone yield depended on the DNA per cell ratio within a very small range.     

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.     

Drug resistance to cis-diamminedichloroplatinum (II) in Chinese hamster ovary cell lines transfected with glutathione S-transferase pi gene

Establishment of Chinese hamster ovary (CHO) cell lines expressing human glutathione S-transferase-pi (GST-pi) was performed after cotransfection of pSV2-neo and human GST-pi cDNA-carrying plasmid p beta actGPi-2. About 30 G418-resistant clones were tested for their expression of GST-pi by Northern blot analysis. Two clones, beta 2-3 and beta 2-5, expressed a significant amount of GST-pi mRNA; and one clone, beta 1-1, that did not was also used for further study. Western blot analysis with anti-GST-pi antibody showed significant increases of GST-pi in beta 2-3 and beta 2-5, but not in beta 1-1. Northern blot analysis with the human GST-pi cDNA probe showed that the increase in the expression of GST-pi-mRNA in beta 2-3 and beta 2-5 was respectively 2- and 4-fold higher than that in beta 1-1. Southern blotting analysis showed that beta 1-1, beta 2-3 and beta 2-5 contained about one copy of the human GST-pi cDNA sequence. beta 2-3 and beta 2-5 were resistant to 1.4- and 3.0-fold higher doses of CDDP than CHO, respectively, but beta 1-1 was not. Increased expression of GST-pi might be associated with CDDP-resistance in CHO cells.     

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.     

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.     

组织专一性表达自杀基因治疗人结肠癌的研究

构建了以癌胚抗原(CEA)基因启动子控制的HSV-TK和ECCD的表达质粒PCEA-TK和pCEA-CD. 将它们分别与pSV2-neo共转染人结肠癌细胞株LoVo和人宫颈癌细胞株HeLa. G418筛选得到细胞克隆LoVo/CEA-TK、toVo/CEA-CD、HeLa/CEA-TK和HeLa/CEA-CD. 与野生型LoVo细胞相比, LoVo/CEA-TK和LoVo/CEA-CD形态无明显改变, 生长曲线也相似, 但对GCV或5-FC的细胞毒的敏感性分别提高了2000倍或700倍.而HeLa/CEA-TK(或HeLa/CEA-CD)仍对低浓度GCV(或5-FC)不敏感. 以上结果显示了应用组织专一性表达的自杀基因治疗人结肠癌的可能性.     

CEA启动子控制HSV—TK基因表达对人结直肠癌细胞的杀伤作用

An expression plasmid pCEA-TK, in which HSV-TK gene was under the control of CEA promoter, was constructed. The human colorectal carcinoma cell line LoVo or the human uterine cervical cancer cell line HeLa was co-transfected with pSV2-neo and pCEATK, respectively. After G418 selection, both transgenic cell clones (LoVo/CEATK and HeLa/CEATK) were obtained. LoVo/CEATK cells were 1300 times more sensitive to the cytotoxicity of ganciclovir than LoVo cells. However, the elevation of GCV sensitivity induced by pCEATK gene in HeLa line was only 8 times. Injection of GCV resulted in significant regression of HSV-TK transfected LoVo tumor in nude mice. These data suggested that the expression of TK gene driven by CEA promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electromicroscopy and DNA fragmentation assay demonstrated that GCV could induce apoptosis in LoVo/CEATK cells. The possibility of the CEATK/GCV system in the treatment of human colorectal carcinoma was discussed.     

The genomic instability associated with integrated simian virus 40 DNA is dependent on the origin of replication and early control region.

DNA rearrangements in the form of deletions and duplications are found within and near integrated simian virus 40 (SV40) DNA in nonpermissive cell lines. We have found that rearrangements also occur frequently with integrated pSV2neo plasmid DNA. pSV2neo contains the entire SV40 control region, including the origin of replication, both promoters, and the enhancer sequences. Linearized plasmid DNA was electroporated into X1, an SV40-transformed mouse cell line that expresses SV40 large T antigen (T Ag) and shows very frequent rearrangements at the SV40 locus, and into LMtk-, a spontaneously transformed mouse cell line that contains no SV40 DNA. Stability was analyzed by subcloning G-418-resistant clones and examining specific DNA fragments for alterations in size. Five independent X1 clones containing pSV2neo DNA were unstable at both the neo locus and the T Ag locus. By contrast, four X1 clones containing mutants of pSV2neo with small deletions in the SV40 core origin and three X1 clones containing a different neo plasmid lacking SV40 sequences were stable at the neo locus, although they were still unstable at the T Ag locus. Surprisingly, five independent LMtk- clones containing pSV2neo DNA were unstable at the neo locus. LMtk- clones containing origin deletion mutants were more stable but were not as stable as the X1 clones containing the same plasmid DNA. We conclude that the SV40 origin of replication and early control region are sufficient viral components for the genomic instability at sites of SV40 integration and that SV40 T Ag is not required.     

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.     

CEA启动子控制HSV-TK基因表达对人结直肠癌细胞的杀伤作用

构建了以CEA启动子控制的HSV-TK基因表达质粒pCEA-TK。转染pCEA-TK的人结肠癌细胞LoVo对GCV的敏感性提高了1300倍。同样条件下,人宫颈癌细胞HeLa对GCV的敏感性仅提高8倍,且对低于血药浓度(20μmol/L)的GCV不敏感。以上结果显示在GCV存在时,CEA启动子控制下HSV-TK基因的表达使CEA阳性的人结直肠癌细胞获得专一性杀伤。此外,DNA片段分析和电镜观察表明GCV诱导转染pCEA-TK的LoVo细胞发生凋亡可能是这个系统杀死肿瘤细胞的机制之一。本工作还讨论了癌胚抗原(CEA)基因启动子用于人结直肠癌专一性自杀基因治疗的可能性。     

Maintenance of expression of differentiated function of kidney cells following transformation by SV40 early region DNA

This study describes the isolation and characterization of epithelial cell lines that maintain their differentiated phenotype following the stable integration of SV40 genes. Epithelial cells were derived from a defined location of rabbit kidney, the thick ascending limb of Henle's loop, and were co-transfected with genes from the early region of SV40 together with pSV2-neo DNA (which confers resistance to the antibiotic G418). These cells were shown to be resistant to G418, express SV40 large T-antigen and continued to express differentiated characteristics typical of cells of their origin. Such characteristics include the expression of high levels of activity of both Na,K-ATPase and the functionally important Na,K,Cl-co-transport system, the synthesis of Tamm-Horsfall glycoprotein and the presence of a barium-sensitive K+ channel on the apical membrane surface.