Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone,Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up, PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgene-expressing cells was 65±10 ng/L per 10~6 cells,420±45 ng/L per 10~6 cells in sense group and 495±30 ng/L per 10~6 cells in the negative controlgroup, (P<0.05), The antisense-VEGF cellclone appeared phenotypically indistinguishablefrom SMMC-7721 cells and SMMC-7721 cellstransfected sense VEGF. The growth rate of theantisense-VEGF cell clone was the same as thecontrol cells. When S. C. was implanted intonude mice, growth of antisense-VEGF cell lineswas greatly inhibited compared with controlcells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.     

Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone.

Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective in XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells of the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure.     

Specific activation in jun-transformed avian fibroblasts of a gene (bkj) related to the avian beta-keratin gene family.

We have analyzed differential gene expression in normal versus jun-transformed avian fibroblasts by using subtracted nucleic acid probes and differential nucleic acid hybridization techniques for the isolation of cDNA clones. One clone corresponded to a gene that was strongly expressed in a previously established quail (Coturnix japonica) embryo fibroblast line (VCD) transformed by a chimeric jun oncogene but whose expression was undetectable in normal quail embryo fibroblasts. Furthermore, the gene was expressed in quail or chicken fibroblast cultures that were freshly transformed by retroviral constructs carrying various viral or cellular jun alleles and in chicken fibroblasts transformed by the avian retrovirus ASV17 carrying the original viral v-jun allele. However, its expression was undetectable in a variety of established avian cell lines or freshly prepared avian fibroblast cultures transformed by other oncogenes or a chemical carcinogen. The nucleotide and deduced amino acid sequences of the cDNA clone were not identical to any sequence entries in the data bases but revealed significant similarities to avian beta-keratin genes; the highest degree of amino acid sequence identity was 63%. The gene, which we termed bkj, may represent a direct or indirect target for jun function.     

Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant.

Although insulin-like growth factor I (IGF-I) is a mitogenic growth factor, its role in tumorigenesis is unclear. We therefore transfected wild-type and truncated beta-subunit mutant (952STOP) human IGF-I receptor cDNAs into Rat-1 fibroblasts. Rat-1 transfectants expressed 2.5- to 7-fold increased IGF-I receptor mass, while the Kd for IGF-I binding was unchanged. The Rat-1 cells transfected with wild-type receptor cDNA responded to in vitro IGF-I treatment by increased proliferation and DNA synthesis. Cells overexpressing wild-type receptors were also transformed as evidenced by ligand-dependent colony proliferation in soft agar. After injection into athymic nude mice, all wild-type transfectants formed solid sarcomas within 3 weeks, and ex vivo tumor cell assays confirmed continued overexpression of human IGF-I receptors. In contrast, both DNA synthesis and proliferation of 952STOP-transfected cells were attenuated below that of untransfected cells. 952STOP cells were nonresponsive to IGF-I in vitro and were unable to sustain anchorage-independent growth. No tumors were induced for up to 8 weeks after injection of 952STOP transfectants into athymic mice, despite the presence of demonstrable endogenous IGF-I receptors on the 952STOP-transfected cells. Therefore, 952STOP behaves as a dominant negative inhibitor of endogenous IGF-I receptor function, probably by assembling nonfunctional hybrid rat/mutant human receptor tetramers.     

Gene therapy for human colorectal carcinoma using promoter controled bacterial ADP-ribosylating toxin genes human CEA: PEA & DTA gene transfer

AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.     

Gene therapy for human colorectal carcinoma using human CEA promoter contro led bacterial ADP-ribosylating toxin genes human CEA: PEA & DTA gene transfer

AIM:To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes. METHODS:Pseudomonas exotoxin A domain [II+III (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa.PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA)promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out. RESULTS:Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION:The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.     

Herpes simplex virus (type 1) thymidine kinase gene does not transform cells morphologically.

BALB/c-derived 10E2 cells were made thymidine kinase(TK)-negative and one isolated clone (B2) was used for studying morphological and biochemical transformations by herpes simplex virus (HSV) type 1 (strain 10412). The B2 cells displayed a "normal" flat appearance and were nontumorigenic in nude mice when tested at frequent intervals over a period of 45 subcultures. B2 cells infected with UV-irradiated HSV (UV-HSV) and maintained in normal growth medium showed foci of spindle-shaped cells after one subculture. The cells from these morphologically transformed foci were tumorigenic in nude mice and were TK negative. B2 cells infected with UV-HSV or transfected with the HSV-1 TK gene and maintained in TK-selective medium showed discrete colonies of cells which displayed a normal flat appearance and expressed the viral TK enzyme. These biochemically transformed B2 cells were nontumorigenic in nude mice. The findings with B2 cells indicate that biochemical and morphological transformations by HSV-1 are independent events and suggest that the HSV-1 TK gene is a suitable vehicle for introducing non-TK genes into cells to assess their transforming potential.     

Retrovirus—mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted. RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.     

Requirement of phospholipase D1 activity in H-RasV12-induced transformation

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.     

Characterization of a porcine genomic clone encoding a major histocompatibility antigen: expression in mouse L cells.

A porcine genomic clone encoding a major histocompatibility, (MHC) antigen was isolated by direct screening of a swine genomic library with a heterologous human MHC cDNA probe. Mouse L cells transformed with DNA from the clone stably express swine MHC antigen. Pig alloantisera specifically lyse transformant but not control cell lines in a complement-mediated cytotoxicity assay. Direct immunoprecipitation of radiolabeled cellular protein from transformed lines by pig alloantiserum results in the coprecipitation of swine MHC heavy chain and mouse beta 2-microglobulin, demonstrating the association of heterologous subunits of MHC antigens.     

Introduction of a human colony stimulating factor-1 gene into a mouse macrophage cell line induces CSF-1 independence but not tumorigenicity

The SV40-immortalized mouse macrophage cell line, BAC1.2F5, is strictly dependent on CSF-1 for its survival and proliferation in culture. Introduction of a retroviral vector containing a 1.6 kilobase (kb) pair human CSF-1 cDNA into these cells abrogated their growth factor dependence but did not render the cells tumorigenic in nude mice. The infected macrophages contained multiple copies of the vector provirus, expressed both membrane-bound and secreted forms of CSF-1, and exhibited constitutive down modulation of the murine CSF-1 receptor. Because insertion of the v-fms gene has previously been shown to abrogate factor dependence and induce tumorigenicity in BAC1.2F5 macrophages, the failure of these cells to express a fully transformed phenotype after persistent stimulation by endogenous CSF-1 suggests that the v-fms and c-fms gene products provide different signals for cell proliferation.     

Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation.

We have introduced the rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector. The ability of the viral DNA to induce foci in the recipient cells was used as a dominant selection marker. Several copies of rat growth hormone DNA were integrated in the mouse cells. The transformed mouse cells expressed rat growth hormone-specific mRNA and secreted mature rat growth hormone. In rat cells, the expression of this gene is regulated by glucocorticoids. We demonstrate that hormone-dependent regulation transfers with the clone and thus appears to be an intrinsic property of the gene or its RNA products.     

Identification of transforming genes of subgroup A and C strains of Herpesvirus saimiri.

Herpesvirus saimiri is an oncogenic herpesvirus that induces rapidly progressing lymphomas in New World primates. Using retrovirus vectors for gene transfer, specific open reading frames of H. saimiri were tested for their ability to transform rodent cells in culture. One open reading frame, designated STP-C488 (for saimiri-transformation-associated protein of the subgroup C strain 488), phenotypically transformed Rat-1 cells, resulting in formation of foci, growth at reduced serum concentration, and growth to higher cell densities. Cells transformed by STP-C488 formed invasive tumors in nude mice. The STP-A11 reading frame of strain 11 (subgroup A) was much less potent in its transforming ability than STP-C488. These results demonstrate the oncogene nature of these two open reading frames and provide a means for studying their transforming functions independent of the rest of the H. saimiri genome.     

Transforming gene in human atherosclerotic plaque DNA.

The monoclonal hypothesis equates atherosclerotic plaques with benign smooth muscle cell tumors and proposes that plaques can arise via mutational or viral events. Here, we provide direct evidence that molecular events, heretofore associated only with tumor cells, are common to plaque cells as well. Three distinct groups of human coronary artery plaque (hCAP) DNA samples transfected into NIH 3T3 cells gave rise to transformed foci. DNA samples from a panel of normal noncancerous human tissues, including coronary artery, were negative in the assay. Southern-blotted focus DNA yielded positive signals when hybridized to the 32P-labeled nick-translated repetitive human "Alu" DNA sequence. The DNA from cloned foci was used successfully in a second round of transfection. Focus DNA hybridized to nick-translated v-Ki-ras, v-Ha-ras, or N-ras probes failed to detect human fragments of these genes. Primary focus cells from each of five clones elicited tumors after injection into nude mice (6/42). Several distinct high molecular weight (greater than 6.6 kilobases) bands were detected after BamHI-digested tumor DNA was hybridized to Alu. Preliminary characterization of these hCAP DNA-associated tumors indicates that they are similar to the fibrosarcomas that arise after injection of ras-transformed cells into nude mice. We propose that transforming genes in plaque cells behave in a manner analogous to the way in which oncogenes behave in cancer cells.     

The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.     

N-myc can cooperate with ras to transform normal cells in culture.

N-myc, a cellular gene bearing homology to the c-myc protooncogene, is frequently amplified and overexpressed in a highly restricted set of related tumors, most notably neuroblastomas and retinoblastomas. We have examined the possibility that N-myc may play a causal role in the genesis of these tumors by defining its ability to transform primary cells in tissue culture. Using an N-myc expression construct capable of producing constitutively deregulated levels of full-length murine N-myc mRNA, we demonstrate that a deregulated N-myc gene can cooperate with the activated Ha-ras oncogene to cause tumorigenic conversion of normal embryonic fibroblasts in a manner indistinguishable from the deregulated c-myc oncogene. Cell lines established from N-myc/ras-transformed foci express high levels of the N-myc gene, and such lines are similar to c-myc/ras transformants in their ability to grow in soft agar and cause tumors in syngeneic rats. These results illustrate that N-myc does encode a c-myc-like transforming activity and that this transforming activity is not specific for the very restricted set of tumors in which N-myc is normally amplified or overexpressed.     

A human oncogene of the RAS superfamily unmasked by expression cDNA cloning.

As an approach to identify human oncogenes, we generated an expression cDNA library from an ovarian carcinoma line. A potent transforming gene was detected by transfection analysis and identified as TC21, a recently cloned member of the RAS gene superfamily. A single point mutation substituting glutamine for leucine at position 72 was shown to be responsible for activation of transforming properties. While the cDNA clone possessed high transforming activity, the ovarian tumor genomic DNA, which contained the mutated TC21 allele, failed to induce transformed foci. Thus, expression cDNA cloning made it possible to identify and isolate a human oncogene that has evaded detection by conventional approaches.