Expression of the transformed phenotype induced by diverse acting viral oncogenes mediates sensitivity to growth suppression induced by caffeic Acid phenethyl ester (cape)

Caffeic acid phenethyl ester (CAPE) displays enhanced growth suppressive and toxic effects toward cloned rat embryo fibroblast (CREF) cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed CREF cells (Su et al, Mol Carcinogen 4: 231-242, 1991). The present study was conducted to determine if transformation of CREF cells with additional oncogenes rendered these cells sensitive to the antiproliferative effect of CAPE. Additionally, studies were conducted to determine if reversion of the transformed phenotype could modify CAPE sensitivity. CAPE displayed increased growth suppressive activity toward CREF cells transformed by a number of oncogenes, including Ha-ras, v-src, v-raf, human papillomavirus type 18 (HPV-18) and human papillomavirus type 51 (HPV-51). Employing Ha-ras-transformed CREF (Ha-ras) and Ha-ras-transformed CREF cells overexpressing the Krev-1 suppressor gene (Ha-ras/Krev-1), evidence for a direct relationship between expression of the transformed phenotype and CAPE sensitivity was demonstrated. Ha-ras/Krev-1 cells displaying a suppression of the transformed phenotype exhibited increased resistance to CAPE-induced growth suppression versus Ha-ras cells, whereas Ha-ras/Krev-1 cells escaping transformation-suppression following in vivo growth in nude mice displayed enhanced sensitivity to growth-suppression induced by CAPE. Similarly, mutant Ad5 (H5hr1)-transformed and v-src-transformed CREF cells displaying a stable reversion in transformation also displayed a reduced sensitivity to CAPE versus their transformed counterparts. These observations indicate a direct relationship between expression of the transformed phenotype and CAPE sensitivity. Elucidation of the mechanism by which CAPE selectively inhibits growth of transformed cells should provide important insights into the critical molecular changes mediating expression of the transformed state and could help identify cellular targets for cancer therapy.     

Immune response to progressor variants derived from transfection of an ultraviolet radiation-induced C3H mouse regressor tumor cell line with activated Harvey-ras oncogene

Skin cancers induced in mice by UV radiation often exhibit a regressor phenotype. In order to determine how tumors escape the immune defenses of the normal immunocompetent host, we sought to isolate progressor variants from a UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select transfected cells. Injection of Ha-ras-transfected UV-2240 cells s.c. into immunocompetent C3H mice produced tumors in four of 36 animals. In contrast, UV-2240 cells transfected with pSV2-neo DNA alone or mock transfected with CaPO4 did not produce tumors in normal C3H mice. DNAs from cell lines established from Ha-ras-induced tumors contained unique Ha-ras sequences in addition to those sequences endogenous to UV-2240 cells. However, the Ha-ras-induced progressor variants did not overexpress the Mr 21,000 protein. The Ha-ra-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. In addition, all four Ha-ras-induced progressor variants produced significantly more experimental lung metastases in nude mice than did the parent UV-2240 cell line. However, both the parental UV-2240 cell line and the Ha-ras-induced progressor variants expressed similar levels of H-2Kk and H-2Dk antigens and were immunologically cross-reactive, as determined by in vitro cytotoxic T-lymphocyte and in vivo immunization-challenge assays. These results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of tumor-specific transplantation or Class I major histocompatibility complex antigens. This implies that some tumor cells can escape the immune defenses of the normal immunocompetent host by mechanisms other than loss of tumor-specific transplantation and Class I major histocompatibility antigens.     

Expression of metastatic potential of tumor cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity

Of 6- and 3-week-old nude mice given intravenous injections of murine tumor cells with well-defined metastatic properties, only the 3-week-old mice developed lung tumor colonies in significant numbers. The quantitative differences in metastatic potential among tumor cell lines injected into syngeneic recipients were also maintained following intravenous injection into young nude mice. Successful metastasis in 3-week-old nude mice is correlated with the low levels of natural killer cell activity detected in these young recipients. Boosting of the natural killer cell activity of 3-week-old nude mice by the administration of bacterial adjuvants and interferon inducers significantly inhibited metastasis formation. The differences in metastasis development could not be attributed to differences in the initial arrest of tumor cells in the pulmonary vascular bed, but rather to a better survival of the arrested cells in the lungs of 3-week-old nude mice as compared with 6-week-old counterparts. We conclude that low levels of NK cell activity are associated with increased incidence of experimental metastasis.     

Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2-E1a

Activated ras oncogene transfection into suitable recipient cells has been shown to induce the metastatic phenotype (Thorgeirsson, et al., Mol. Cell. Biol., 5: 259-262, 1985). We have used this model system to study the correlation of basement membrane collagenolysis with metastatic propensity. The c-Ha-ras oncogene alone, or combined with v-myc, transfected into early passage rat embryo fibroblasts, induce these cells to secrete high levels of type IV collagenolytic metalloproteinase and to concomitantly exhibit a high incidence of spontaneous metastases in nude mice. Cotransfection of c-Ha-ras plus the adenovirus type 2 E1a gene yields cells which are highly tumorigenic but nonmetastatic and fail to produce type IV collagenase. This effect is due to a suppression of collagenase elaboration, not increased production of a collagenase inhibitor, and not decreased production of a collagenase activator. The characteristics of the collagenase are identical to tumor type IV collagenase described previously. The nonmetastatic cells which failed to produce type IV collagenase retain the ability to secrete high levels of plasminogen activator. Transfection with the protooncogenic forms of Ha-ras or mos, or spontaneous transformation of NIH 3T3 cells or chemical transformation of BALB 3T3 cells yields cells which fail to produce collagenase, are tumorigenic, but totally nonmetastatic. These data support a biochemical linkage of type IV collagenase expression with the metastatic phenotype in this rodent system.     

Synergistic Suppressive Effect of Double Transfection of Tumor Necrosis Factor-α and Interleukin 12 Genes on Tumorigenicity of Meth-A Cells

Tumor necrosis factor-α(TNF-α) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer/lymphocyteactivated killer (NK/LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK/LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK/LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-α and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.     

Synergistic suppressive effect of double transfection of tumor necrosis factor-alpha and interleukin 12 genes on tumorigenicity of Meth-A cells.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer / lymphocyte-activated killer (NK / LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK / LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK / LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-alpha and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.     

Interleukin 6 gene transfection into Lewis lung carcinoma tumor cells suppresses the malignant phenotype and confers immunotherapeutic competence against parental metastatic cells.

To investigate the influence of interleukin 6 (IL-6) production on malignancy of tumor cells we transfected cells of the high-metastatic, low-immunogenic D122 clone of the Lewis lung carcinoma with a mammalian expression vector containing the human IL-6 complementary DNA. In vitro, IL-6 positive transfectants showed growth inhibition that was directly correlated with the levels of IL-6 production. The in vitro growth arrest did not seem to be a function of an autocrine system mediated via the secreted human IL-6 acting on the tumor cell surface receptors since neutralizing antibodies to human IL-6 did not prevent the growth inhibition. Neither did exogenous human recombinant IL-6 affect the growth of D122 cells. In vivo, IL-6 positive transfectants showed reduction of tumorigenicity and significant suppression of metastatic competence in syngeneic, immunocompetent mice. In mature T-cell deficient nude mice, the IL-6 transfectants showed some arrest of local growth but no suppression of lung metastasis. It seems therefore that the reduction of metastatic competence of IL-6 transfectants is primarily a function of stimulation by the transfectants of host T-cell immune responses. Immunization with inactivated high-positive IL-6 transfectants induced high levels of anti-tumor cytotoxic T-lymphocytes and protected mice against metastatic growth of a subsequent graft of parental tumor cells. Moreover, reduction of metastatic growth of parental highly metastatic D122 cells was also achieved when immunization of mice was begun after establishment of the primary parental tumors. Thus, inactivated IL-6 transfectants were effective when used as a cellular vaccine for experimental immunotherapy of metastasis.     

Transformation of normal homologous cells by a spontaneously activated Ha-ras oncogene

Several tumor-derived oncogenes have been shown to independently act as complete carcinogens following transfection into target cells from established tissue culture lines. However, the number and types of oncogenes required to transform primary cultures of normal mammalian cells is unclear. To clarify this issue in a simplified model system, we transfected genomic DNA from a naturally occurring rat tumor into NIH/3T3 cells as well as into early passage rat embryo fibroblasts. The 3T3 cells were transformed with high efficiency to malignant phenotypes; the rat embryo cells were transformed at lower frequencies following cotransfection with a selectable neomycin resistance marker and treatment with Geneticin (G418). The transformed rat cells had cancerous phenotypes as determined by in vitro, cytogenetic, and in vivo criteria. Moreover, the transformed mouse and rat cells contained new tumor DNA-derived nucleotide sequences homologous to the activated human Ha-ras oncogene. Elevated levels of Ha-ras-specific mRNA, as well as enhanced expression of the Mr 21,000 oncogene product, were detected in the transformed cells. Therefore, under well-defined experimental conditions, a spontaneously activated Ha-ras oncogene from a naturally occurring tumor was able to independently transform normal, homologous cells to a malignant phenotype.     

Metastatic conversion of factor-dependent lung fibroblasts (CCL39) requires over-expression of oncogenes which induce high rate of autonomous replication

Several classes of oncogenes were tested for their ability to confer cellular growth autonomy and the metastatic phenotype on CCL39 lung fibroblasts. v-sis as well as highly but not weakly expressed activated ras, v-fps and myc genes were susceptible to relieve CCL39 cells from their dependence on exogenous growth factors. However, based on growth rate estimations, ras and fps cells divided 2 to 3 times more rapidly than myc and sis cells in serum-free medium. All ras and fps cells produced pulmonary metastases in 60-100% of young nude mice, following subcutaneous or intravenous injection. Acquisition of factor-independent growth during in vivo passage was demonstrated in two instances. Animals developed either no or sporadic metastases after implantation of transfected cells expressing v-sis, normal Ha-ras, myc or no foreign oncogene. The results are consistent with the notion that the rate at which tumor cells can proliferate independently from growth factor stimulation is a good predictor of their metastatic potential. Oncogenes such as activated ras and fps appear more efficient than myc and sis to induce the metastatic conversion of preneoplastic CCL39 cells and to abrogate Go-arrest controls of division.     

ras-transformed NIH 3T3 cell lines, selected for metastatic ability in chick embryos, have increased proportions of p21-expressing cells and are metastatic in nude mice

We previously reported that ras-transformed NIH 3T3 cells could be selected in vivo for increased metastatic ability after intravenous injection into chick embryos, and that the metastatic cell populations expressed increased levels of ras p21 protein. We have tested the metastatic ability of a series of these cells in nude mice, to determine if their properties in the chick embryo experimental metastasis assay predict their metastatic behavior in nude mice. We report here that cells selected for metastatic ability in the chick embryo are also metastatic when assayed in nude mice. We also asked whether the selected cells uniformly expressed higher levels of p21, using an immunocytochemical procedure. We found that p21 expression among individual cells in these populations was quite heterogeneous. There was a direct relationship between the proportions of p21-expressing cells and metastatic ability in both assays, with increased proportions of p21-expressing cells in cell lines selected for metastatic ability. Our results suggest that (a) the experimental (i.v.) metastasis assay in the chick embryo offers an efficient and cost-effective procedure for the identification and selection of cells that are also experimentally metastatic in nude mice, and (b) the metastatic properties of ras-transformed NIH 3T3 cells are due to individual cells that express increased amounts of p21.     

In vivo optical imaging of human adenoid cystic carcinoma cell metastasis

A noninvasive, whole-body, real-time fluorescence optical imaging of stable high-level green fluorescent protein (GFP)-expressing human adenoid cystic carcinoma (ACC-M-GFP) was demonstrated for in vivo visualization of metastatic behavior in nude mice. Five-week-old female nude mice were injected with ACC-M-GFP in the primary organ: submandibular gland. Metastases were only visualized by GFP expression in the lung. However, metastatic lesions of ACC-M-GFP in the lung, muscle, bladder and bony were found by imaging of GFP expression in intact mice through tail vein injection of ACC-M-GFP cells. The construction of highly fluorescent and stable GFP transfectants of ACC-M has revealed the multi-organ metastatic capability of ACC-M cells through this optical imaging.     

Therapy of murine mammary carcinoma metastasis with interferon gamma and MHC gene-transduced tumour cells.

Gene-transfected tumour cells were used to cure mice bearing lung metastases by the parental, non-transduced mammary adenocarcinoma (TSA-pc). Repeated subcutaneous (s.c.) administrations of mitomycin C (MitC)-treated interferon gamma (IFN-gamma) transfectants induced a 90% inhibition in the number of lung metastases. Therapeutic effect required an intact T-cell response, as shown by the lack of efficacy in nude mice. Autocrine stimulation by IFN-gamma induces specific modifications in the phenotype of transfectants that acquire a high metastatic ability and show a high expression of IFN-responsive genes; these two features were exploited to design two experimental protocols to obtain an improvement of the therapeutic effect. The increased metastatic ability of IFN-gamma transfectants was used to deliver IFN-gamma selectively to the lungs of mice bearing TSA-pc pulmonary metastases. A significant therapeutic effect was obtained when TSA-pc experimental metastases were treated by repeated intravenous (i.v.) injections of MitC IFN-gamma transfectants. Since i.v. administrations of IFN-gamma transfectants did not induce immune memory, the therapeutical effect appeared to depend on the inflammatory-like response activated by local IFN release. To exploit the autocrine stimulation of IFN-sensitive genes an IFN-gamma transfectant clone was subjected to a second transfection with an allogeneic class I MHC gene (H-2K(b) or H-2D(h)). IFN-gamma plus MHC double transfectants maintained IFN-gamma release, showed a very high expression of the MHC gene products, stimulated both macrophages and T cells, and were less tumorigenic in immunocompetent mice than the parent IFN-gamma clone. Therapeutic efficacy of double transfectant IFN-gamma plus H-2D(b) cells against TSA-pc was superior to single transfectants, showing that the reaction elicited by genetically engineered cells can be selectively tuned to increase therapeutic efficacy.     

Enhancement of metastatic potential by gamma-interferon

Preincubation of murine colon 26 colon adenocarcinoma cells with gamma-interferon (IFN-gamma), but not alpha-interferon, produced a significant increase in experimental pulmonary metastases in syngeneic BALB/c and T-cell-deficient BALB/c nude mice. The enhancement was seen after as little as 1 h of exposure to 1 unit/ml of IFN-gamma and persisted for at least 72 h following removal of the cytokine. IFN-gamma exerted its effects by increasing the pulmonary retention of cells during the first 6 h following tumor cell injection. During this period all cells visualized in the lung were trapped in pulmonary capillaries. The enhancement was not due to modulations in class I major histocompatibility complex surface antigen expression; nor was it due to alterations in cell size, adhesion to components of the extracellular matrix in vitro, heterotypic or homotypic adhesion, sensitivity to lysis by activated peritoneal macrophages, osmotic fragility, enhancement of surface class II major histocompatibility complex antigen expression, or enhancement of intercellular adhesion molecule-1 (ICAM-1). Colon 26 was completely resistant to natural killer cell-mediated lysis in vitro, and IFN-gamma did not modulate the ability of colon 26 to form conjugates with isolated splenocytes. In vivo elimination of anti-asialo GM1 + cells increased pulmonary metastasis, and in such mice, there was no longer a difference in metastatic potential between control and IFN-gamma-treated cells. We conclude that low doses of IFN-gamma generated at the site of the tumor by host-infiltrating cells or during cytokine therapy could enhance the survival of tumor cells in the circulation and enhance their metastatic potential.     

Follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice.

PURPOSE: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell-depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. EXPERIMENTAL DESIGN: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. RESULTS: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell-depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. CONCLUSIONS: These results suggest that FST plays a critical role in the production of multiple-organ metastasis, predominantly by inhibiting the angiogenesis. This is the first report to show the role of FST in metastases.     

The role of the H-ras oncogene in radiation resistance and metastasis

The sensitivity of tumor cells to the killing effects of ionizing radiation is thought to be one of the major determinants of curability of tumors in patients treated with radiation therapy. This paper reviews the evidence from our laboratory and other groups which supports a role for oncogenes in the induction of radioresistance in cultured mammalian cells. Primary rat embryo cells (REC) were chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offered several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of synergism between oncogenes on the induction of the radioresistant phenotype. The results show that the activated H-ras oncogene induces radiation resistance in primary rat cells after transformation, but that the effect of the oncogene itself is small. However, the myc oncogene, which has no effect on radiation resistance by itself, appears to have a synergistic effect on the induction of radiation resistance by H-ras. Radiation resistance induced by H-ras plus myc is characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. The AdenoE1A oncogene which will also act synergistically with ras in transformation assays plays a less clear-cut role in assays of radiation resistance. The H-ras oncogene is also known not only to transform cells but also to induce metastatic behavior in the tumors which form after these transformed cells are injected into syngeneic animals or nude mice. We have also shown in our primary rat embryo cell system that the induction of metastatic behavior in transformed cells, like the induction of radioresistance depends on a complex interaction between oncogenes and the cellular background. This evidence will be reviewed to demonstrate some of the analogies between radiation resistance and metastasis as examples of the complex alterations in cellular phenotype which occur after oncogene transfection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the Ha-ras (RasH) oncogene in mediating progression of the tumor cell phenotype (review)

Recent experimental evidence indicates that the c-Ha-ras (rasH) oncogene may be causally involved in the etiology and evolution of specific human neoplasms. In addition, cultured cells transformed by the rasH oncogene can induce both a tumorigenic and a metastatic phenotype when expressed in appropriate cultured cells. To begin to define the molecular and biochemical mechanism(s) by which the rasH oncogene induce their effects on expression of the transformed state we have employed a cloned rat embryo fibroblast (CREF) cell line. Transformation of CREF cells with wild-type 5 adenovirus (Wt) results in transformed cells which display anchorage-independence and an increased saturation density in monolayer culture, but are non-tumorigenic in both athymic nude mice and syngeneic Fischer rats. In contrast, when CREF cells are transformed with mutant type 5 adenoviruses, such as H5hrl, or the ElA transforming gene from hrl (0-4.5), tumors are induced in both nude mice and syngeneic rats. However, hrl (0-4.5)-transformed CREF cells are not metastatic following intravenous injection into the tail vein of syngeneic rats. Insertion of an activated T24 rasH oncogene or a wild-type v-rasH oncogene into CREF, wt-transformed CREF or hrl (0-4.5)-transformed CREF cells results in acquisition of a metastatic phenotype by these cells. A mutant v-rasH oncogene (mutant 116K), which is defective in GTP binding and the induction of transformation of NIH 3T3 cells, does not induce transformation in CREF cells, but it can progress wt-transformed CREF cells to a tumorigenic-non-metastatic state. Employing this model system which displays well-defined and stable stages in the tumor cell progression lineage, we have analyzed the potential role of changes in the phosphatidylinositol (PI) cycle and phospholipase A2 (PLA2) enzyme activity during progression to a tumorigenic and metastatic phenotype. An increase in PI cycle intermediates (primarily inositol triphosphate; IP3) were observed only in the wt-transformed and hrl (0-4.5)-transformed CREF cell lines transfected with the rasH oncogene. In the case of PLA2, all rasH-transformed CREF cell lines displayed increased activity. In contrast, CREF cells transformed only by Ad5 (Wt or hrl (0-4.5)) or the 116K v-rasH oncogene did not display increased PLA2 activity similar to that observed in rasH transfected cells. Since one important metabolite generated by PLA2 is arachidonic acid, which is converted into prostaglandins and leukotrienes by cyclooxygenase or lipooxygenase, respectively, the levels of prostaglandin E2 (PGE2) in the various cell lines were monitored.(ABSTRACT TRUNCATED AT 400 WORDS)