Oncogene-mediated human lung epithelial cell transformation produces adenocarcinoma phenotypes in vivo

It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets.     

ENU-induced in vitro neoplastic transformation of rat mammary epithelial cells.

Normal mammary epithelial cells, originating from female Sprague-Dawley rats, were grown in Dulbecco's Modified Eagles Medium containing 25% horse serum and hormone supplements. Once established as an epithelial cell culture, the cells were treated with N-ethyl-N-nitrosourea (ENU) in various doses (25-500 ug/ml) to study the process of in vitro mammary epithelial cell neoplastic transformation. The ENU-treatment of primary mammary epithelial cell culture resulted in a sequence of phenotypic changes, termed stages I-V. The rat mammary epithelial cells, after a period of approximately 30 days post-ENU exposure, showed a marked proliferation of morphologically altered cells which formed multi-layered colonies. Subsequently, these cells acquired the capacity to form colonies in soft agar and eventually produced a high yield of palpable tumors when inoculated into newborn female isologous hosts or female athymic nude mice. The immediate effect of ENU on these cells was monitored by measurement of cellular DNA content, unscheduled DNA synthesis, cell proliferation and chromosomal aberrations. The ENU effect on cell proliferation and DNA synthesis was dose dependent; doses greater than 100 ug/ml reduced the cell number and DNA synthesis. Cytofluorometric histograms of non-ENU-treated rat mammary epithelial cells showed a near diploid population of cells. The ENU exposed cells subsequently became hyperdiploid (24-72 hours after ENU) and then regained their near diploid pattern at 120 hours after ENU exposure. The ENU-treated cells also showed a second peak of cells with DNA content in the tetraploid and octaploid range at 24-72 hours after ENU exposure. Single chromatid breaks, isochromatid breaks, chromosomal exchanges, multiple chromosomal breaks and double minutes were among the chromosomal aberrations seen in ENU-treated cells. Most of the chromosomal aberrations peaked at 6 hours post-ENU exposure. The ENU-induced model of in vitro meplastic transformation of rat mammary epithelium as described in this communication appears to provide a good model for the systematic study of the early critical cellular events prerequisite to this carcinogenic process.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

Surface structure of fetal rat brain cells during neoplastic transformation in cell culture.

The surface microstructure of fetal rat brain cells undergoing neoplastic transformation in long-term cell culture after a single transplacental pulse of 75 microgram N-ethyl-N-nitrosourea/g body weight to the fetal (18th day of gestation) BD IX rat was investigated by scanning electron microscopy. After about 3 weeks of culture, N-ethyl-N-nitrosourea-pretreated fetal rat brain cells showed focal proliferation of neural cells on an underlayer of flat, epithelioid cells. The neural cells exhibited varying forms of numerous dorsal ruffles and an increased number of other surface microprojections. Between the 40th and the 100th day, nodules of bipolar and multipolar neural cells were observed with a complex surface microstructure including many blebs and ruffles and an increased number of microvilli. After 100-210 days, more rapidly proliferating, morphologically altered cells formed "piled-up" foci, which resulted in a homogeneous population of cells with numerous long microvilli, large ruffles, and blebs over the whole surface. The cells retained the same altered surface structure until tumorigenicity after reimplantation into the syngeneic host was first observed (approximately 273 days). Surface alterations characteristic of the neoplastic cells were thus observable more than 100 days before the cells became tumorigenic.     

Transmission electron microscopy of fetal rat brain cells during neoplastic transformation in cell culture.

Fetal rat brain cells were investigated by transmission electron microscopy during neoplastic transformation in long-term cell culture. Before transfer of the cells to culture, BD IX rat fetuses were treated with a single transplacental pulse of N-ethyl-N-nitrosourea (75 micrograms/g body wt) on the 18th day of gestation. During the early stages (3--4 mo), both glia-like and neuron-like cells were present in the culture, and after 2 months they formed complex aggregates ("nodules"). In contrast, corresponding secondary control cultures consisted of flat, epithelioid neural cells without neuron or astrocyte differentiation. After 3 months, cells with neuron morphology gradually disappeared. Some of the remaining cells contained many autophagosomes. After 5 months, rapid proliferation of rather homogeneous, glia-like populations was accompanied by reduction of microfilament bundles and microtubules, as well as atypical nuclei. Ability to form tumors upon sc implantation into syngeneic hosts was not observed until about 3 months later.     

Neoplastic transformation of fetal rat brain cells in culture after exposure to ethylnitrosourea in vivo.

A single, transplacental pulse of N-ethyl-N-nitrosourea (ENU; 75 mug/g body wt) to the fetal (18th day of gestation) BD IX rat led to death with malignant tumors of the central and peripheral nervous system after a median time of approximately 195 days. In contrast to untreated control cells, dissociated brain cells transferred to long-term cell culture 20-90 hours after the ENU pulse became tumorigenic after approximately 200 days, as assayed by reimplantation into baby BD IX rats. This was preceded by a characteristic sequence of phenotypic alterations (termed "stages I-IV"). During early primary culture (stage I), both ENU and control cultures exhibited stationary glia-like cells on a growing layer of epithelioid (possibly glia precursors) and few fibroblast-like cells. Stage II (approximately 10th-40th day) was characterized by a constant proportion of glia-like cells in the ENU cultures and by their disappearance in the controls. During stage III (approximately 40th-100th day), slowly proliferating glia-like cells in the ENU cultures formed "piled-up" foci. They could then be removed from the underlying cell layer and cultured separately. Transition to stage IV (approximately 100th-200th day) was marked by proliferation of morphologically altered cells, which subsequently acquired the capacity of form colonies in semisolid agar and finally became tumorigenic (stage V). This system may represent a model for the study of malignant transformation.     

Induction of transplantation immunity to rat colon carcinoma isografts by implantation of intact fetal colon tissue

The objective of this study was to demonstrate whether intact fetal colon tissue can induce immunity to large-bowel cancer isografts. Adult rats were immunized against fetal antigens by implantation of mitomycin-C-treated intact fetal colon tissue beneath the renal capsule. Control animals received implants of pieces of intact adult colon tissue. Kidneys containing the implants were removed after 3 or 5 weeks and the rats were challenged with isografts of a 1,2-dimethylhydrazin (DMH)-induced colon carcinoma. Significant inhibition of tumor growth was observed in immunized groups compared to controls. The present results indicate that some embryo-fetal antigens expressed in intact colon tissue of 13 to 15-day-old fetuses are also expressed in colon carcinoma cells and can act as transplantation antigens.     

Gene expression in clonally derived cell lines produced by in vitro transformation of rat fetal hepatocytes: isolation of cell lines which retain liver-specific markers.

The pattern of gene expression in fetal hepatocytes transformed in culture with a hepatocarcinogen (FRL cells) is studied with respect to a range of markers which are either developmentally regulated and/or shown to be expressed at high levels in hepatoma cells. The relative abundance of the respective mRNAs is determined and immunocytochemistry is used to detect the respective proteins in cultured cells. When compared with its normal counterpart, FRL cells retain the expression of transferrin, alpha 1-acid glycoprotein, gamma-glutamyltranspeptidase, and tyrosine aminotransferase at near normal levels, while expression of the liver-specific isoenzymes of pyruvate kinase (L form) and aldolase (B form) is reduced. The cell lines are different in that they fail to express albumin, alpha-fetoprotein, thiostatin and alpha 2-macroglobulin, and they express high levels of M2-pyruvate kinase and aldolase A, markers often found in abundance in hepatoma cells. Therefore transformation has resulted in different effects on different genes. Furthermore, it is of interest to find that the cells coexpress both forms of the pyruvate kinase isoenzymes which does not occur in the normal developing hepatocyte. These results indicate that it is possible to use this model to study changes which accompany transformation of fetal rat hepatocytes. The resulting cell lines have a stable phenotype and retain the changes which result from transformation even after extended passaging. This facilitates comparisons between the precursor cell and the tumor cell, both of which can be maintained under controlled conditions which exist in culture.     

Promoting effect of 12-O-tetradecanoylphorbol-13-acetate on the in vitro malignant transformation of fetal rat brain cells exposed in utero to ethylnitrosourea

In order to investigate the possibility that the theory of two-stage carcinogenesis can be applied to neurogenic carcinogenesis, we analyzed the promoting effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the in vitro malignant transformation of fetal rat brain cells exposed in utero to ethylnitrosourea (ENU). Rat brain cells were transferred to a cultured system at 72 h after a single pulse of ENU (50 mg/kg body weight) to pregnant SD-JCL rats on the 18th day of gestation. The positive findings of glial fibrillary acidic protein and S-100 protein in primary cultured cells by the analysis of immunohistochemistry revealed the neuroglial origin of transformed cells. These cells were divided into 12 groups and were treated twice per week with or without TPA at concentrations from 0.1 to 50.0 ng/ml. From the results of cellular morphology, Concanavalin A agglutinability, colony forming capacity in semisolid soft agar, and tumorigenicity in vivo, malignant transformation of fetal rat brain cells appeared earlier in the ENU group treated with TPA than in the untreated ENU group. On the basis of these observations, it is suggested that TPA might be effective as a tumor promoter on ENU-induced neurogenic carcinogenesis.     

In vitro transformation of rat esophageal epithelial cells with N-nitrosobenzylmethylamine

Using an explant/cell culture system, rat esophageal epithelialcells were transformed in vitro by exposure to N-nitroso-N-benzyl-N-methylamine(BMNA). Twelve esophageal explant cultures per group were exposedtwice (at days 1 and 7) to 0.0, 2.5, 5.0 or 10.0 µg BMNA/mlof medium. After incubation for 60–90 days, epithelialcells in primary cultures treated with all three concentrationsof BMNA could be subcultured and cell lines were developed.The number of primary cultures and the number of subsequentlydeveloped epithelial cell lines was carcinogen-dose-dependent.Cell lines could only be established from carcinogen treatedexplants. Electron microscopy revealed that the BMNA-treatedcell lines contained morphological markers of esophageal epithelialcells; i.e., numerous tonofilaments and junctional complexes,even after prolonged subculture. By immunofluorescence, thecells reacted positively with antibodies prepared to mouse skinprekeratins (K₁ and K₂). Two cell lines (from the 5 µgBMNA/ml group) were able to grow in soft agar and produce palpabletumors upon injection into syngeneic recipients. These tumorspossessed the histological features of squamous cell carcinomas.     

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.     

In vitro transformation induces abnormal protein thiol levels in rat liver cells

Cells from an established line of normal rat hepatocytes (IAR 20) were irradiated with 3 Gy gamma-radiation from a cobalt source to generate transformed clones. Cells from four transformed colonies were compared with the parent cell line by flow cytometry following staining with ortho-phthalaldehyde and propidium iodide to quantitate protein thiols and DNA respectively. Transformed cells exhibited an increased variability in cellular protein thiols which was most evident in G1 and S phase. The altered pattern of macromolecular thiol distribution implies early changes in redox state and/or modification of the amounts or types of sulphur-containing proteins in transformed cells.     

Oncogene-mediated regulation of p53 ISGylation and functions

Oncogene-mediated cellular transformation is a multistep process involving activation of growth-promoting pathways as well as inactivation of tumor suppressors. We recently found that ISGylation of the p53 tumor suppressor is an important novel mechanism to control its stability. Here we identified that Isg15-dependent regulation of p53 can be enhanced by different oncogenes. We further show that the Src-mediated phosphorylation of p53 on Tyr126 and Tyr220 has a positive effect on p53 ISGylation by enhancing Herc5 binding. In turn, deletion of Isg15 results in accumulation and activation of native p53 in transformed cells thus increasing its anti-cancer activity and suppressing tumorigenesis in mice. We propose that Isg15-dependent degradation of p53 is an alternative pathway for oncogenes to regulate p53 activity, and thus is an attractive pathway for development of new anti-cancer drugs.     

Effect of alkyl-lysophospholipid on glioblastoma cell invasion into fetal rat brain tissue in vitro

The antitumor effect of alkyl-lysophospholipid (ALP) was studied on a continuous glioma cell line (GaMg) as well as on tumor spheroids obtained from three different primary brain tumor biopsies. GaMg monolayer growth was reduced by 50% after treatment with 30 microM ALP; cells accumulated in the G2M phase of the cell cycle as determined by flow-cytometric analyses. Tumor spheroid growth was reduced by 25 and 44% during treatment with 10 and 30 microM ALP, respectively. These drug concentrations also caused a severe destruction of spheroids. No effect on growth or morphology was seen in spheroids treated with 0.1 and 1.0 microM ALP. ALP caused a dose-dependent inhibition of invasion by GaMg tumor spheroids into brain aggregates. After 168 h of 1.0 microM ALP treatment, the volume of the intact brain aggregate was 90% larger than that in the untreated co-cultures. To further investigate the efficacy of ALP as an anti-invasive drug, co-cultures were performed with specimens obtained from three primary brain tumors: a highly invasive glioblastoma multiforme, an anaplastic astrocytoma, and an astrocytoma. Treatment of spheroids from the most invasive tumor with ALP caused a 7-fold preservation of normal brain tissue relative to control co-cultures. Moreover, the sensitivity of primary glioma spheroids to the anti-invasive effect of ALP seemed to be associated with the aggressiveness of the tumor; spheroids from the more malignant specimen (glioblastoma multiforme) were more sensitive than those from the less aggressive tumors. The anti-invasive effect seen with nontoxic concentrations of ALP may prove valuable in the treatment of malignant gliomas.