Clonal analysis of tumorigenicity and paratumorigenic phenotypes in rat liver epithelial cells chemically transformed in vitro

Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemical, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DNA content; growth rate in culture; activities of gamma-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and therefore is not reliable as a "marker" phenotype for neoplastic transformation in cultured rat liver epithelial cells. The poor correlations, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and progression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isogeneic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.     

Actin filaments and tumorigenicity in a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet-irradiated HSV

The characteristics of the cytoskeleton of a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet (UV)-irradiated HSV were studied by indirect immunofluorescence using anti-actin IgG. Parental 3Y1 cells possessed well-developed actin filaments, while 3Y1 cells transformed by UV-irradiated HSV also retained well-developed actin filaments. Transformed cells were divided into 2 groups according to tumorigenicity in newborn Fischer rats; one had a strongly tumorigenic potential and the other a weakly tumorigenic potential. Tumor-derived cell lines exhibited a highly tumorigenic potential, and were also divided into 2 groups, one with well-developed actin filaments and the other without well-developed actin filaments. Our results suggested that transformation or tumor formation by HSV is a multi-step process and that morphological loss of actin filaments in the cells is not essential to the tumorigenic potential of the cells transformed by HSV.     

Human cells transformed in vitro by human cytomegalovirus: tumorigenicity in athymic nude mice.

Athymic nude mice were inoculated with human embryo lung cells transformed in vitro by human cytomegalovirus (CMV). Of the inoculated animals, 62% developed tumors after an average latent period of 19 days. The tumors were composed of small, polygonal cells with large nulei and scanty cytoplasm embedded in an abundant collagenous matrix. The cells were poorly differentiated but may have been of epithelial origin. Adjacent structures were rarely invaded. CMV-related intracellular and membrane antigens were detected by indirect and anticomplement immunofluorescence techniques in cells cultured in vitro from the tumors.     

Assay of the tumorigenicity of transformed cells on the chorio-allantoic membrane of chick embryo

Assay of the tumorigenicity of transformed cells on the chorio-allantoic membrane of fowl eggs Monodispersed suspensions of transformed cells from tissue cultures produce tumors following inoculation on the chorio-allantoic membrane of fowl eggs. This capacity (tumorigenicity or transplantability on the CAM) can be assayed quantitatively by counting tumors produced in 7 days on 11-day-old eggs. The tumor counts are log-normally distributed and mean counts are proportional to the number of inoculated cells when they are low (less than 10). The error decreases with the increase in the number of eggs and 95% confidence intervals less than 0.48 log₁₀ have been obtained in series of over 10 eggs. Assays are reproducible; the mean number of cells required to produce one tumor (TD) generally remained constant in established clonal lines of transformed cells, which suggests that it is a stable genetic character of the cells. However, in some lines, the TD decreased to a lower plateau, presumably by selection of cell variants with increased transplantability. The TD of established transformed rodent cells was generally lower than that of transformed primate cells with a limited life span; one line of morphologically transformed human cells even produced no tumors. Tumors have also been obtained with morphologically untransformed, but established BHK21/13 hamster cells, but not with untransformed unestablished diploid primate and fowl cells. The TD of transformed and untransformed BHK21 cells is higher than, but proportional to, the TD₅₀ of the same cells in Syrian hamsters.     

Differential tumorigenicity of 3T3 cells transformed in vitro with polyoma virus and in vivo selection for high tumorigenicity

BALB/c 3T3 cells transformed in vitro with a temperature-sensitive mutant of polyoma virus were cloned. Forty-eight clones examined demonstrated heterogeneity with respect to doubling-time in vitro and tumorigenicity in syngeneic mice in vivo. Observation periods that lasted in certain cases as long as 2 years showed that some clones exhibited a relatively high tumorigenicity, i.e., they yielded a relatively high incidence of tumors following a small inoculum of cells and a relatively short latency period. Other clones were relatively low tumorigenic: even high tumor cell inocula yielded a relatively low tumor incidence following a relatively long latency period. These results indicate that at least in this system variation in tumorigenicity is generated independently of host factors. An intraclonal heterogeneity with respect to the length of the precancer latency period was seen. Some tumors appeared relatively early following inoculation of cloned cells, whereas others appeared considerably later following an identical inoculum of the same clone. Cloned in vitro transformed cells were passaged once in syngeneic mice and recultured. The single in vivo passage cycle augmented considerably the tumorigenicity of these cells as compared to their in vitro maintained clonal ancestors. The increased tumorigenicity of the in vivo passaged cells is due, most probably, to the in vivo induction and/or selection of high tumorigenic intraclonal variants. The survival time of mice bearing high tumorigenicity variants was very similar to that of mice bearing low tumorigenicity variants.     

Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.     

The tumorigenicity of 5-azacytidine in the male Fischer rat

5-Azacytidine was administered to young adult male Fischer rats.Tumors were found in 31 out of 70 rats that had received 5-azacytidineand survived 18 months from the start of the experiment. Severalrats had multiple primary tumors. In the rats that were testedfor complete carcinogenicity a variety of tumor types was found.These included acute leukemia and malignant reticuloendotheliosis,and tumors of the testis, skin, and bronchus. No hepatic tumorswere found in the group that was tested for hepatic tumor initiation.Hepatocellular carcinomas were found only in the group thatwas examined for hepatic tumor promotion by receiving a priorinitiating dose of diethylnitrosamine. No tumors were foundin the age controls. Thus, in these initial experiments, 5-azacytidineappeared to be a complete carcinogen, inducing tumors in severalorgans, and a tumor promoter but not a complete carcinogen forthe liver.     

Lack of correlation between transformed characteristics in culture and tumorigenicity of mouse mammary tumour cells

Androgen responsive and unresponsive Shionogi 115 mouse mammary carcinoma cells have been examined for anchorage-independent growth and tumorigenicity in nude mice. The two cell types exhibit transformed and normal growth characteristics respectively, but both give rise to tumours in nude mice. No correlation between tumorigenicity and transformed characteristics including anchorage-in-dependent growth could be demonstrated.     

Induction of apoptosis by retinoids in human cervical-carcinoma cell-lines

Retinoids can inhibit the growth and modulate the differentiation of a variety of tumor cell types in vitro and in vivo. All-trans retinoic acid (ATRA) and N-(4-hydroxyphenyl)retinamide (4HPR) are currently being evaluated in clinical trials for their potential use in cancer chemoprevention and therapy. We compared the effects of these retinoids on 10 human cervical carcinoma cell lines. Four of the 10 cell lines showed dramatic morphological changes and the other 5 exhibited decreased cell density after treatment with 10 mu M 4HPR, whereas few changes were induced by 10 mu M ATRA. Cell rounding and detachment were also observed in four of the cell lines. An analysis of DNA from both detached and attached cells after retinoid treatment has demonstrated the formation of a DNA ladder after electrophoresis in agarose gels, which indicated that some of the cell lines had undergone apoptosis. Induction of DNA fragmentation by 4HPR but not by other retinoids (ATRA, 13-cis-RA, and 9-cis-RA) was further evidenced as early as 24 h after treatment by a quantitative assay based on the degradation of [H-3]-thymidine-labeled DNA. Ln addition, morphological changes of nuclei associated with apoptosis such as chromatin condensation were observed by propidium iodide staining of the nuclei after 4HPR treatment. These results demonstrate that 4HPR causes apoptosis in several cervical carcinoma cell lines and that it is more potent in this effect than ATRA or other RA isomers.     

Multiple loci on human chromosome 11 control tumorigenicity of BK virus transformed cells

BK virus (BKV) is a human papovavirus that readily transforms rodent cells, but not human cells, to a neoplastic phenotype, suggesting that tumor-suppressor functions expressed in human cells control BKV oncogenicity. Transfer of a normal human chromosome 11 to BKV-transformed mouse cells suppresses the malignant phenotype. In this report we map the regions of chromosome involved in tumor suppression. Transfer of chromosome 11 to the BKV-transformed hamster cell line HKBK produces monochromosomic hybrids retaining only portions of the transferred human chromosome. We have compared the tumorigenicity of the hybrids with the molecular mapping of chromosome 11 retained regions. This analysis indicated that 3 regions of human chromosome 11, 11p15.5, 11p13 and 11q13, cooperate in tumor suppression. However, 11q13 seems the most important, since all the HKBK/H11-induced tumors analysed had lost this region, whereas 11p15.5 and 11p13 were sometimes retained. The chromosomal regions identified in this study are deleted in several types of human tumors, suggesting that the BKV transformation system specifically detects tumor-suppressor genes on chromosome 11 that are involved in human oncogenesis. This model may be of use in isolating and cloning such genes. The results of this report raise the possibility that BKV may have a synergistic tumorigenic effect in human cells where tumor-suppressor genes controlling its oncogenk potential are inactivated. © 1994 Wiley-Liss, Inc.     

Establishment and in vivo characterization of multidrug-resistant dunning R3327 rat prostate-carcinoma cell-lines

We describe the selection of 3 new multidrug-resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma. Cell lines of weak (AT2) and strong (AT3 and MAT-LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations. Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay). Expression of the multidrug-resistance glycoprotein (P-170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219. The behavior of the parental and resultant drug-resistant cells was assessed by their growth in syngeneic rats. Doxorubicin challenge of the initially drug-sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P-glycoprotein. While lung and lymph-node metastases developed in host animals inoculated with parental AT3 and MAT-LyLu cells, no metastases developed in the multidrug-resistant progeny of these cell lines. This study has shown that Dunning rat prostate-carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug-resistant and express P-glycoprotein following exposure to doxorubicin. Further more, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential. Nevertheless, it is unlikely that the non-metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P-glycoprotein. These new multidrug-resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable mode with which to analyze the relationship between expression of P-glycoprotein and the metastatic phenotype of prostatic carcinoma cells.     

Enhanced tumorigenicity of fibroblasts transformed with human herpesvirus 8 chemokine receptor vGPCR by successive passage in nude and immunocompetent mice

The human herpes virus 8 (HHV-8)-encoded G protein-coupled chemokine receptor (vGPCR) has been implicated in the pathogenesis of Kaposi's sarcoma (KS), particularly because of its high constitutive signaling activity. Here, we used retroviral transduction to generate vGPCR-expressing 3T3 fibroblasts that are tumorigenic in nude mice, but as expected fail to induce tumors in their immunocompetent counterparts. However, tumor fragments obtained from nude mice grow progressively in immunocompetent BALB/c mice. Unexpectedly, vGPCR-expressing cells established from grafted tumor fragments gave rise to tumors in immunocompetent mice. These tumors exhibit a striking histological resemblance to KS including plump spindle cell morphology, a high degree of vascularization and brisk mitotic activity. High expression of vGPCR was confirmed in the cell lines and tumors using a newly developed vGPCR-specific monoclonal antibody. Finally, short interfering RNA directed at vGPCR abrogated or significantly delayed tumorigenesis in mice, demonstrating that the tumor development is specifically driven by vGPCR. This novel model for vGPCR-mediated oncogenesis will contribute to our understanding of the role of vGPCR in the pathogenesis of HHV-8 and may even be important in identifying critical molecular and epigenetic changes during tumor progression in vivo.     

Suppressed autophosphorylation and tyrosine kinase-activity in revertant cell-lines expressing rat Neu oncogene

The revertants of neu oncogene-transformed cell lines, neu-R1 and neu-R2, have been shown previously to be phenotypically and morphologically nontransformed, yet both expressed the rat neu oncogene. In both cell lines the rat neu lacked significant tyrosine phosphorylation. We show that the intrinsic tyrosine kinase activity of neu-encoded p185 protein of both revertants was suppressed as well, and the p185 neu protein contained extracellular conformational changes that may inhibit receptor activity. Five other revertant cell lines that lost neu upon reversion were transfected with a rat neu oncogenic construct. They remained nontransforned but expressed the oncogene. Like neu-R1 and neu-R2, the transfectants expressed p185 that lacked receptor tyrosine phosphorylation. The inability of neu to induce transformation in these cell lines may be through a common mechanism that suppresses receptor autophosphorylation and tyrosine kinase activity.     

Demonstration of oncogenic potential of mammalian cells transformed by DNA-containing viruses following photodynamic inactivation.

The oncogenic properties of hamster embryo cells transformed by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and SV40 virus following photodynamic inactivation using neutral red were determined by subcutaneous inoculation into newborn Syrian hamsters. Cells transformed by all three viruses produced palpable tumors after different latent periods. Histopathological examination showed that HSV-2 tumors were fibrosarcomas and metastases were often seen in the lungs. HSV-2 primary tumors were reinoculated subcutaneously into weanling hamsters; they developed palpable tumors within 2 weeks. HSV-specific antigens were detected in the cytoplasm and/or on the surface of both the HSV-1 and HSV-2 tumor-cell cultures by the indirect immunofluorescence technique. The same method revealed SV40 tumor antigen in the nuclei of the SV40 tumor cells. Sera from HSV or SV40 tumor-bearing hamsters gave positive reactions when tested against HSV-infected hamster cells or SV40-infected monkey cells, respectively. These results demonstrate that herpes simplex virus and SV40, whose infectivity was lost following photodynamic inactivation, retained the virus genetic information necessary for transformation of normal cells to an oncogenic phenotype.     

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40 The soluble components of different cell lines in various species (hamster, mouse, rat and dog) were compared by means of precipitation test in agar. Some lines were spontaneously transformed, others were transformed by SV40 virus and oncogenic viruses. The utilization of rabbit and hamster antisera, specific for a clone of SV40 transformed cells (TSV₅Cl₂) demonstrated the presence of three new antigens in the various cell strains transformed by SV40. These antigens are different from the nuclear T antigen and probably also from the transplantation antigen located on the cell membrane.