Tumour-induced host stromal-cell transformation: induction of mouse spindle-cell fibrosarcoma not mediated by gene transfer

Tumour-induced host-cell transformation has been addressed by examining human tumours in situ and following xenograft to nude mice. We have found evidence for the transformation of host stromal fibroblasts both in vivo and following the introduction of the tumours to in vitro culture. The in vitro culture of one such xenograft--derived from a human prostatic adenocarcinoma--resulted in the outgrowth of a transformed aneuploid mouse cell line. This transformed line was tumourigenic both in BALB/c nu/nu (nude) mice and in heterozygous nu/+mice, with the morphology of a spindle-cell sarcoma. The cell line did not express human isozymes or human histocompatibility antigens, nor were human chromosomes present. Moreover, human DNA sequences were not detected by human Alu repeat sequence element probing in the transformed cell line grown either in vitro or in vivo. The line contained retroviral long terminal repeat sequences but there was no evidence of proviral activation. These findings indicate that tumour cells may cause transformation of neighbouring stromal cells; that this transformation may proceed in the absence of DNA transfer or activation of endogenous proviruses; and that the means of this observed transformation may involve humoral factors elaborated by the tumour cells.     

p53/T-antigen complex disruption in T-antigen transformed NIH3T3 fibroblasts exposed to oxidative stress: correlation with the appearance of a Fas/APO-1/CD95 dependent, caspase independent, necrotic pathway

Simian Virus 40 Large T-antigen expressed in NIH3T3 cells increases p53 level and interacts with this tumor suppressor to form large nuclear complexes. We show here that T-antigen sensitizes NIH3T3 cells to low doses of the oxidative stress inducer menadione. This oxidant increased p53 accumulation and disrupted p53/T-antigen interaction, but not T-antigen/pRb, T-antigen/Hsc70 and p53/Hsc70 complexes; a phenomenon inhibited by the anti-oxidant N-acetyl-cysteine. Analysis of several p53 downstream gene products revealed that the level of Fas receptor, which was sharply reduced by T-antigen expression, was drastically increased in response to menadione treatment. Menadione also induced a T-antigen dependent cleavage of Fas ligand. Analysis performed with Fas receptor antagonist antibody and metalloproteinases inhibitor revealed that menadione triggers a Fas-dependent death of a fraction of T-antigen expressing cells. This Fas pathway does not activate caspase 8 or 3, probably because of the inhibition induced by T-antigen, and leads to a necrotic cell death which contributes at least in part to the hypersensitivity of T-antigen transformed cells to oxidative stress.     

Loss of the oncogene from human H-ras-1-transfected NIH/3T3 cells grown in the presence of excess methionine

Mouse NIH/3T3 cells, transfected with T24 genomic DNA (J10 cells), carry the human H-ras-1 (hu-H-ras-1) oncogene stably integrated in the chromosomal DNA and display the transformed phenotype with a "criss-cross" morphology and capacity for anchorage-independent growth. When these cells were cultured in the presence of 25 mM DL-methionine, they lost their transformed phenotype; the characters of the normal phenotype (before transfection) reappeared, even though cell viability, as measured by the cloning potential on a solid support, was fully conserved. Southern blot analysis showed that cells continuously propagated in methionine-supplemented medium (J10met), unlike cells grown in normal medium, progressively segregated the hu-H-ras-1 gene. The progressively fewer cells still retaining the oncogene could be selected by inoculation of J10met cells into nude (nu/nu) mice of Swiss background. After prolonged methionine treatment, however, no such oncogene-positive cells could be detected, even when the excess methionine was withdrawn from the culture medium after 105 days. Conclusive evidence for the loss of the hu-H-ras-1 oncogene sequence was provided by subcloning the J10met cells. The oncogene sequence could no longer be found, and a fraction of subclones was no longer tumorigenic when assayed in nude mice. Other subclones generated late tumors negative for the hu-H-ras-1 transforming sequence, and a small fraction of subclones gave rise to rapidly growing early tumors that were also negative for the transforming sequence. The question of how methionine can induce the phenotypic and genetic change from malignancy to normality is discussed.     

Oncogenic potential of human herpesvirus-6 DNA

The ability of human herpesvirus-6 (HHV-6, GS strain) DNA to neoplastically transform established NIH3T3 cells was examined. Transfection of NIH3T3 cells with the intact genomic DNA (170 Kb) or the subgenomic clone pZVH14 containing an 8.7 Kb insert followed by focal or G418 selection resulted in the formation of morphologically transformed cells. When injected subcutaneously into nude mice, these cell lines gave rise to rapidly growing tumors while cells transfected with control vector DNA did not grow in agarose and did not produce tumors in mice. By Southern blot analysis, HHV-6 DNA was detected in the G418 selected primary transfectants and tumor cell lines, but not in the focus derived transformed and tumor cell lines. The data suggest that HHV-6 DNA contains sequences which may contribute to neoplastic transformation, but are not likely to be required for maintenance of transformation.     

An alternative assay system for the detection of transforming genes

We have developed an alternative assay system based on DNA mediatedgene transfer (DMGT) for the detection of functional transforminggenes which we find more sensitive and reliable than other assaysystems. This approach involves selecting in tissue culturefor cells which are expressing a drug-resistance marker co-transferredwith the tumour DNA thus isolating the small number of cellswhich have taken up and are expressing exogenously added DNA.The ability of transformed cells present in this drug-resistantcell population to form tumours in athymic (nu/nu) nude micewas then used to assay for transforming genes. Using NIH3T3and Rat 2 cells as recipients, tumours produced after DMGT withDNA from non-tumorgenic cells were only very rarely observed.Tumour induction was observed using DNA from a biopsy of a humanovary carcinoma and a spontaneous murine lung carcinoma. Twohuman retinoblastoma cell lines were negative for tumour induction.The transforming gene from the ovarian carcinoma was identifiedas c-K-ras2.     

Transformation of rabbit kidney cells by BKV(MM) human papovavirus.

Primary rabbit kidney cells were transformed by BKV(MM), a papovavirus isolated from the urine of a child with the Wiskott-Aldrich syndrome. The transformed cells contained BK T-antigen, but no antigen that reacted with SV40 U-antiserum. The transformed cells failed to produce tumors in nude mice, and BKV (MM) was not rescued from transformed cells by cell fusion or chemical induction methods. The transformed cells supported the growth of rabbit kidney vacuolating virus (RKV), and could be used to quantitate RKV by plaque formation under an agar overlay.     

Cellular and molecular changes in 3T3 cells transformed spontaneously or by DNA transfection

Transformed cell lines have been selected following exposure of NIH 3T3 cells to a calcium phosphate precipitate containing DNA from: the human colon carcinoma cell line, SW 480; the cloned Harvey sarcoma virus ras gene; the parental NIH 3T3 cell line; or no DNA (a spontaneous transformant). Unlike the parental 3T3 cells, each of these lines readily formed malignant spindle cell tumors in Swiss nu/mu mice. Southern blots confirmed the presence of the human homologue of the Kirsten ras gene in the cells transformed by SW480 DNA, and the Harvey ras gene in the cells transformed with that cloned sequence. The morphology of each of the lines was different, the cells transformed with the human and viral ras genes being the most aberrant (but not identical) and forming the most extensive foci in culture. These ras transformed lines also exhibited anchorage-independent growth, while the two other transformed lines did not. Both of the ras transformed lines, as well as the spontaneously transformed line, exhibited a pronounced disruption of actin microfilament structure. Two-dimensional gel electrophoresis of 35S-methionine-labeled peptides revealed that these three lines also had a marked decrease in an acidic peptide of 32K daltons.     

Thy-1 as a negative growth regulator in ras-transformed mouse fibroblasts.

To identify molecules on the cell surface involved in negative growth regulation, we assumed that their amounts would be reduced after malignant transformation. We analyzed several proteins by fluorescence-activated cell sorter in mouse NIH 3T3 and its transformed cell lines. Surprisingly, the amount of Thy-1, a cell surface glycoprotein anchored in the cell membrane by a glycophosphatidyl inositol linkage, was significantly decreased in the transformed NIH 3T3 lines, especially in ras-transformed NIH 3T3 lines. The malignant properties of clones of NIH 3T3 transformed by Kirsten murine sarcoma virus have a good correlation not only with the high amount of RAS proteins but also inversely with the amount of Thy-1. NIH 3T3 subpopulations lacking Thy-1 exhibit more susceptibility to the induction of colony-forming ability in soft agar by Kirsten murine sarcoma virus than the Thy-1-positive populations. Finally the transfection of Thy-1 complementary DNA to the ras-transformed NIH 3T3 significantly inhibits the colony formation in soft agar as well as the tumor formation in nude mice. Our results suggest that Thy-1 has negative effects on the anchorage-independent growth of ras-transformed NIH 3T3 cells.     

DNA transformation activity of a human gastrocarcinoma cell line

DNAs of three cell lines of human gastrocarcinoma (MGC-803, BGC-823 and PACM-82) and two fresh solid tumors of human stomach cancer were used to transfect NIH3T3 and Rat-1 cells. The transformed cells were selected with high concentration of glucose and low concentration of serum, or with medium containing Geneticin (G418) after co-transfection of pSVneo and DNAs of stomach cancer cell line or primary transformants. From the second round transfection, we had obtained transformants which could grow with high colony forming efficiency in soft agarose and were tumorigenic in nude mice. The southern blot analysis showed that the cellular DNA of the transformants contained human Alu repeat sequence and the transformed gene from stomach cancer cell line (BGC-823) and was homologous to proto-oncogene c-Ha-ras. The transforming gene is able to induce neoplastic transformation of NIH3T3 and Rat-1 cells.     

Comparison of nude mice with the host species for evaluation of the tumorigenicity of guinea pig and hamster cells transformed in vitro by chemical carcinogens.

Nude mice (nu/nu) were compared with the species of origin for determination of the tumorigenicity of cells from chemical carcinogen-transformed and noncarcinogenic chemically treated, nontransformed guinea pig and Syrian hamster cultures. The incidence and time of appearance of progressively growing tumors were similar in the host species and in nude mice after injection of 10(7) transformed cells. Inoculation of 10(8) nontransformed cells routinely was nontumorigenic in the host species and in nude mice. The nude mouse has potential as a sensitive and reliable alternative host to the species of origin to elaluate the tumorigenicity of xenogeneic mammalian cells from cell culture model systems of carcinogenesis.     

The relationship between metastatic phenotype and steady expression of BGC-Ha-ras oncogene from metastasis cell lines in nude mice (abstract)]

NIH/3T3 cells transformed by activated BGC-Ha-ras (6.6 kb) with a point mutation at codon 12 were able to induce tumor in nude mice with lung metastasis. The metastatic phenotype seemed stable in vivo metastasis assay. After two round subculture of the successively induced metastasis foci, two cell lines, GCM-1/3T3 and GCM-2/3T3, were established. In Southern blot analysis it was found that the bands from GCM-1/3T3 and GCM-2/3T3 were the same. Based on Southern analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RPLF), it was proved that the activated c-Ha-ras (6.6 kb) existed all along in the genomes of the transformed and metastatic culture cells. Amplification and over-expression of activated c-Ha-ras were shown by DNA and RNA dot blot hybridization in transformed and metastatic culture cells. The metastatic phenotype might be related to the existence and steady expression of the point mutated ras.     

Selection for spontaneous metastasis after calcium phosphate-mediated transfer of DNA

The ras oncogene has been shown to induce metastatic potential in a wide variety of cells. However, one cell line, C127, when transformed by ras (HC127), proved to be an exception in that the transformed cells gained the ability to form tumors in nude mice, but these tumors did not form spontaneous metastases. Because C127 cells do not metastasize after ras transfection, these cells can be used to identify other factors which contribute to the development of metastatic potential. Specifically, these cells can be used as recipients in DNA transfer experiments utilizing DNA from other cells in which H-ras has been used to induce the metastatic phenotype, thus allowing the search for genes in addition to ras itself which may be necessary for metastasis. A system utilizing genomic DNA transfer into C127 cells transformed by ras has been developed. These cells were used as the recipient for genomic DNA in cotransfection with pSV2neo followed by selection both in the experimental i.v. assay and in the spontaneous metastasis assay in nude mice. DNA from two lines with metastatic potential (both transformed by ras genes) gave rise to metastatic clones, whereas DNA from the recipient cells, HC127, NIH 3T3 cells, and two human tumor cell lines, CHP126 and A2058, failed to give rise to transfectants which could metastasize. The metastases after the first cycle were put into culture, and DNA was extracted from these cells and used in a second cycle of transfection. The capacity to metastasize was also transferred in the second cycle. Of seven metastatic clones examined, four showed no detectable rearrangements of the transfected v-H-ras gene, but in three of these clones, there were rearrangements of this gene, which was originally present in the recipient cell, HC127. This indicates that in a subset of the selected clones there may have been selection for rearrangements of the host genome rather than introduction of foreign DNA sequences, and that such effects must be considered in gene transfer experiments. It is also possible that the transfer of particular genomic DNAs are leading to genetic instability in these experiments. mRNA levels were compared in the metastatic variants and the parental cells; H-ras-specific RNA was raised from 4- to 22-fold in the metastatic cell lines, regardless of rearrangement of the v-H-ras gene.     

Induction of tumors in Syrian hamsters by a human renal papovavirus, RF strain.

Injection of RF virus (RFV), a papovavirus isolated from human urine, into newborn Syrian hamsters induced subcutaneous sarcomas in 50% of the recipients with 18- to 48-week latent periods. Transplantation of 2 X 10(6) primary RFV-induced tumor cells into weaning hamsters caused tumors in 100% of the recipients within 1-2 weeks. Continuous tissue culture cell lines were established from two primary tumors; one of these was transplantable. An in vitro-transformed continuous cell line (RF-194) obtained by infection of primary hamster embryo fibroblasts with RFV was transplantable in weaning hamsters. Neither infectious RFV nor virion antigens were detected in transformed cells. No RFV was recovered when transformed cells were fused with permissive, human embryo kidney cells by means of inactivated Sendai virus. Immunoperoxidase staining was used to show that all three RFV-transformed cell lines contained an intranuclear T-antigen closely similar to that of simian virus 40(SV40)-infected cells. Most hamsters (84%) with primary or transplanted RFV tumors responded with antibodies that reacted with RFV T-antigen and the T-antigen of SV40-infected cells. Likewise, hamster antisera against SV40 T-antigen cross-reacted with RFV T-antigen. Adsorption of RFV T-antisera with an excess of lyophilized SV40-transformed cells removed all detectable activity against SV40 T-antigen but left significant activity against RFV T-antigen. The reciprocal adsorption produced an antiserum spedicic for SV40 T-antigen. Thus human and simian papovavirus T-antigens were related but immunologically separable.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.     

Constitutive overexpression of a 89 kDa heat shock protein gene in the HBL100 human mammary cell line converted to a tumorigenic phenotype by the EJ/T24 Harvey-ras oncogene

The non tumorigenic human mammary cell line HBL100 has been transformed by the EJ/T24 human bladder carcinoma Harvey(Ha)-ras oncogene. Six cell lines were established from transformed colonies. They all expressed a high level of the ras oncogene and were tumorigenic in athymic nude mice. During an in vivo passage in animals, tumour cells presenting a growth advantage were selected, and some of the tumours revealed an amplification of the transfected ras sequences. Using this model of human cell transformation, we have isolated a cDNA clone corresponding to a heat shock protein gene (hsp89 alpha). This gene, normally transcribed at a higher rate in response to serum stimulation, was found to be constitutively overexpressed in ras-transformed HBL100 cells. In contrast, a closely related hsp gene (hsp89 beta), remained sensitive to serum stimulation, in both untransformed and ras-transformed HBL100 cells. Thus, the regulation of the expression of the hsp89 genes, upon serum stimulation, involves ras-dependent and ras-independent pathways. Constitutive overexpression of the murine homolog of the hsp89 alpha was observed in NIH3T3 cells transformed by the three ras oncogenes, but not with some other oncogenes. Therefore, alteration of the hsp89 alpha gene expression is not a general characteristic of transformed cells, but seems to be linked to ras transformation.     

Retinoid-specific induction of differentiation and reduction of the DNA synthesis rate and tumor-forming ability of a stem cell line from a rat mammary tumor

Differentiation of the stem cell line rat mammary (Rama) 25 to alveolar-like cells can be monitored by the increase in production of domes (hemispheric blisters) in the cell monolayer and immunoreactive casein in the tissue culture medium. This step was accelerated not only by the synthetic inducer dimethyl sulfoxide (DMSO) but also by all-trans-retinol, all-trans-retinal, all-trans-retinoic acid (RA), and all-trans-retinyl acetate (concentration range, 0.04-4 microM) in the presence of the hormones prolactin, hydrocortisone (HC), insulin, and 17 beta-estradiol; 9-cis-all-trans-retinal was without effect. A combination of RA and HC was active in producing doming, whereas RA, all four hormones, and serum were required for maximum production of immunoreactive casein. The retinoids in the same concentration range also caused a reduction in the DNA synthetic rate in a similar time period. When Rama 25 cells were treated with RA and the four hormones yielding the droplet and doming cultures, subsequent injection of these cells into young, female inbred nu/nu (nude) mice led to a reduced incidence of tumors compared with injections of untreated cells. Tumorigenic variant cell lines were selected previously from Rama 25 that were either elongated and failed to differentiate at all to doming and casein-secreting cultures (Rama 521) or that did so spontaneously but whose rates were not accelerated by addition of DMSO (Rama 259). Both Rama 521 and Rama 259 failed to respond to the retinoids and hormones in producing domes and immunoreactive casein, in decreasing DNA synthetic rates, and in lowering the incidence of tumors induced by injection of the cell lines into nude mice. Thus the anticancer activity of the retinoids in rat mammary gland carcinogenesis may be due in part to their differentiation-inducing properties.     

The state of immortalized, transforming and suppressor genes in rat-cells, transfected by polyomavirus large-T and hpv18 e6+e7 genes

Several novel cell clones (A1-A6) derived from the rat embryo fibroblasts immortalized by the polyoma virus T-antigen (LT) gene and transformed by HPV18 E6 and E7 genes were explored. Using E6 or E7 peptide antisera we detected E6 and E7 proteins with approximate molecular masses of 16 kDa and 20 kDa, respectively. Monoclonal antibody to p53 PAb421 but not PAb240 precipitated different but appreciable amounts of p53 protein in all cell clones, indicating that wild-type p53 is expressed in these cells. So expression of HPV18 E6 protein in cells does not always lead to a complete reduction in p53 levels. The quantity of p53 protein in cell clones did not correlate with the level of polyoma virus large T-antigen expression. Variations in levels of p53 protein in clones did not influence on such cell biological properties as anchorage-independence and tumorigenicity for nude mice which were similar in all cell clones.