Gap junction expression following UVC-induced neoplastic transformation in human hybrid cell lines

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC- irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC- induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.      

Transformation renders MDR cells more sensitive to polyunsaturated fatty acids

A series of genetically related cell lines that express mdr genes but differ in their ability to form tumors has been challenged with gamma-linolenate and eicosapentaenoate to verify if the sensitivity of tumorigenic mdr cells to cytotoxic PUFAs differs from the sensitivity of non-tumorigenic mdr cells. The tumorigenic mdr cell lines were derived by transformation of their parental non-tumorigenic mdr cell line with myc and ras oncogenes. Four ras and five myc transformed cell lines were used for the estimation of clonal variability. The data as based on colony forming assays, showed that six out of nine of the tumorigenic mdr cell lines were more sensitive than the non-tumorigenic mdr cells. These results suggest that a tumorigenic phenotype renders mdr cells more sensitive to PUFAs and that PUFA supplementation either alone or in conjunction with existing forms of cancer therapy may have significant clinical implications.     

Monoclonal antibodies directed against rat tracheal epithelial cells transformed in vitro

Cloned hybridoma cell lines were obtained by fusion of murine myeloma cells with spleen lymphocytes of either F344 rats or BALB/c mice immunized against the malignant F344 tracheal cell line, 2-10-1. Monoclonal antibodies were selected for their ability to bind to the immunizing cell line and not to normal tracheal epithelial cells. Results from quantitative binding assays indicated that such monoclonal antibodies recognized six epitopes. Each epitope was detected on four other malignant tracheal epithelial cell lines, and was also expressed during early preneoplastic cell passages (i.e. before the cells acquired the ability to produce carcinomas in vivo). Quantitative differences in epitope expression between non-tumorigenic and tumorigenic passages of individual cell lines could not be detected for five of the epitope groups. However, two monoclonal antibodies, recognizing the same epitope, did show quantitative binding differences between non-tumorigenic and tumorigenic cell passages on three of the five cell lines tested. Our results show that carcinogen-altered tracheal cell populations can be distinguished from non-altered cells (by our current assay methods) by use of monoclonal antibodies. The suggest that the antigen expression is an early event associated with the transformation of rat tracheal epithelial cells in culture.     

Processing of three different types of DNA damage in cell lines of a cutaneous squamous cell carcinoma progression model

In order to study the role of DNA damage processing in the development of cutaneous squamous cell carcinoma (SCC), we assessed the ability of six keratinocyte cell lines from a multistage-tumor progression model to repair three types of DNA damage: pyrimidine dimers, oxidative DNA lesions and DNA double strand breaks (DSB). The model comprised the spontaneously immortalized, non-tumorigenic human keratinocyte cell line HaCaT, four different c-Ha-ras transfectants of HaCaT (non-, benign- and two malignant-tumorigenic) and a SCC-derived cell line. Host cell reactivation assays with UVB-treated plasmid vectors pRSVcat showed no significantly altered repair of UVB-induced pyrimidine dimers in the tumorigenic cell lines, compared with the non-tumorigenic lines. Using the singlet oxygen-treated plasmids pRSVcat the Ha-ras-HaCaT- clones and the SCC-cells, exerted a DNA repair efficiency that was not significantly different from HaCaT cells. In order to assess the ability of the cells to ligate free DNA ends (repair of DSB), we used a plasmid shuttle vector assay with linearized plasmid pZ189. We found a significant increase of DNA end joining ability in the non-tumorigenic, the benign and in one of the malignant HaCaT-clones II-4. The malignant HaCaT-clone II-3, however, exerted a significantly lower rate of rejoining the linearized plasmid. This cell line also showed a highly and significantly elevated rate of micronuclei, which reflects a pronounced chromosomal instability. The SCC-cells exhibited a more efficient repair of DNA DSB than the HaCaT cells. We conclude that in the examined model, progression of human keratinocytes from the non- tumorigenic to the highly tumorigenic phenotype, is not accompanied by a decrease in the cell's capacity to repair UVB- and singlet oxygen- induced DNA lesions. However, an acquired deficiency in repairing DNA double strand breaks can be one mechanism promoting progression towards malignancy, possibly through impairing chromosomal stability.      

Altered dexamethasone responsiveness and loss of growth control in tumorigenic mouse lung cell lines

Glucocorticoid hormones induce differentiation, inhibit proliferation, and, in mice, reduce carcinogen-induced tumorigenesis of lung epithelial cells. Therefore we examined dexamethasone effects on tumorigenic and non-tumorigenic mouse lung epithelial-derived cell lines. Non-tumorigenic cells were growth inhibited and exhibited CAT activity in pMMTV-CAT transfectants in response to dexamethasone. Tumorigenic cell lines exhibited a range of responses to dexamethasone. While one tumorigenic line was growth-inhibited and responsive in CAT assays, 2 other tumorigenic cell lines were unresponsive both in CAT and in growth assays. A fourth tumorigenic cell line exhibited intermediate sensitivity in CAT assays and was actually growth-enhanced by dexamethasone. Although no difference between cell lines was observed in the abundance of glucocorticoid receptor protein on Western blots, the least dexamethasone-responsive tumorigenic lines exhibited very little binding of ³H-dexamethasone. Clones of turnorigenic lines stably transfected with the rat glucocorticoid receptor gene were more dexarnethasone-sensitive in CAT assays and were growthinhibited by dexamethasone. These data suggest that the neoplastic progression of cell lines derived from mouse lung frequently involves the acquisition of diminished glucocorticoid responsiveness.     

Analysis of oncogene, tumor suppressor gene, and chromosomal alterations in HeLa x osteosarcoma somatic cell hybrids.

Using a series of tumorigenic and non-tumorigenic somatic cell hybrids that resulted from the fusion of the human osteosarcoma cell line OHS50-P16T (P16T) with the HeLa cell line D98OR, we investigated the role that genetic mutations, including alterations of oncogenes, tumor suppressor genes, and chromosomes, play in P16T tumorigenicity. Analysis of a previously identified oncogene mutation, c-myc amplification, in the P16T cell line demonstrated that both the tumorigenic and non-tumorigenic hybrids contained the amplified c-myc gene. Analysis of previously identified P16T tumor suppressor gene alterations, p53 mutation, and loss of RB1 expression demonstrated that the mutated p53 gene was selectively maintained in both the non-tumorigenic and tumorigenic hybrids, whereas loss of RB1 expression was not maintained in either the non-tumorigenic or tumorigenic hybrids. Chromosomes 11, 13, 17, and 22 were analyzed for loss of heterozygosity (LOH) to characterize the status of these previously described chromosomal alterations in the tumorigenic and non-tumorigenic hybrids. Loss of HeLa D98OR chromosome 22, with maintenance of P16T chromosome 22, was observed in the tumorigenic hybrids, a result confirmed by LOH analysis, which demonstrated the specific loss of HeLa chromosome 22 genetic material in the tumorigenic segregants. Together, these results demonstrated that amplified c-myc, mutant p53, and RB1 genes seem to be important in osteosarcoma tumorigenicity and that an additional altered gene or genes on chromosome 22 may play a key role in osteosarcoma tumorigenicity.     

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.     

Expression of gamma-glutamyltranspeptidase in cultures of spontaneously and chemically transformed rat liver cells

The expression of gamma-glutamyltranspeptidase (gamma-GTP) and the relationship between this enzyme activity and tumorigenicity were studied in 38 tumorigenic and non-tumorigenic epithelial cell lines derived from livers of normal and aminoazo-dye-fed rats. Nine of 22 non-tumorigenic epithelial cell lines exhibited gamma-GTP-positive cells, whereas 9 of 16 tumorigenic epithelial cell lines did not exhibit gamma-GTP-positive cells. This finding revealed that no correlation existed between the acquisition of gamma-GTP activity and tumorigenicity. None of 10 spontaneously transformed epithelial cell lines, which consisted of 8 tumorigenic and 2 non-tumorigenic cell lines, exhibited gamma-GTP-positive cells. On the other hand, 16 of 28 transformed epithelial cell lines, which were derived from aminoazo-dye-fed rat livers and aminoazo-dye-treated cultures of normal rat liver cells, included gamma-GTP-positive cells in various percentages. As described above, in contrast to in vitro spontaneous transformation of rat liver cells, it is obvious that the carcinogen-induced transformation of rat liver cells in vitro as well as in vivo was frequently accompanied by the acquisition of gamma-GTP-activity. Therefore, it is concluded that the expression of gamma-GTP in liver cells may be mainly associated with exposure to chemical carcinogens.     

Changes in DNA content of rat tracheal epithelial cells during neo-plastic progression in vitro

Flow cytometric DNA analysis was used to compare nuclear DNAcontent of carcinogen-induced F-344 rat tracheal epithelialcell lines as they progressed from non-tumorigenic (preneoplastic)to tumorigenic (neoplastic) populations in vitro. Normal trachealcell populations were used as diploid reference cells. All ofthe tracheal epithelial cell lines established from carcinogen-treatedtracheas showed increases in nuclear DNA content as comparedto normal cell populations. For five cell lines, measurementswere made during the preneoplastic state as well as after conversionto the neoplastic state. Four of the five cell lines showeda major shift in DNA content as the culture progressed frompreneoplastic to neoplastic populations. However, there wasno consistent change in DNA content as cultures progressed toneoplastic populations in vitro. Two cell lines showed shiftsto higher levels as the cultures became tumorigenic, while twoshowed shifts to lower levels. Additionally two cell lines (3F3and 165D) had DNA distribution profiles indicative of mixedcell populations during the neoplastic phase. Cloning experimentsof cell line 3F3 confirmed that those cells having a model DNAvalue the same as that of their preneoplastic progenitor populationswere non-tumorigenic. Evidence that such shifts in DNA contentcorrelate with comparable changes in chromosome number was presentedfor the 3F3 cell line. These studies demonstrate that the transitionfrom preneoplastic tracheal epithelial cells to neoplastic populationsis often associated with a change in DNA content, and wouldsuggest that the malignant cell type emerges as a new cell typefrom preneoplastic progenitor populations.     

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.     

The plate-implant as a bioassay for the neoplastic potential of cultured cells

One objective of this study was to test the plate implant as an in vivo bioassay for the neoplastic state with pairs of cell lines that were non-tumorigenic and tumorigenic when inoculated in suspension at various sites. Each pair originated from a common cell pool or from one cell derived from normal rat or mouse embryonic tissue. The implantation of cells attached to polycarbonate plates was compared with the subcutaneous injection of comparable numbers of cells in suspension or the intraocular injection of 10⁵ cells. In addtiion, some implants of glass helices with attached cells were made into syngeneic mice and compared with injection of comparable cell numbers intramuscularly into X-irradiated hosts. A total of 10 lines or clones were tested on plates. Of four lines that were non-tumorigenic when inoculated in suspension either subcutaneously or intraocularly, two were tumorigenic when implanted subcutaneously attached to plastic plates. The remaining two are the first permanent rodent lines that have not been tumorigenic on plastic plates. Of the remaining six lines, all were tumorigenic on plates and two produced sarcomas at a higher frequency on plates than in suspension. Cytologic diagnoses of the cells implanted on plates, subcutaneously and intraocularly, correlated best with the results from plate implants. Of three mouse lines implanted on helices, two were equally tumorigenic in suspension and on helices, whereas the third was tumorigenic on helices only. The plate implant was more satisfactory technically than the helices and proved to be one of the most sensitive bioassays for the neoplastic state of cultured cells.     

Mutation rates and mutational spectra in tumorigenic cell lines

The rate and molecular nature of spontaneous mutations at the hgprt locus were examined in a series of tumorigenic and non-tumorigenic closely related Chinese hamster fibroblastic (CHEF) cell lines. Mutation rates of tumorigenic cells determined by fluctuation analysis were found to range from the low rate also seen in non-tumorigenic cells to values increased up to 25-fold. No simple correlation was found between elevated mutation rates and tumorigenic potential. The nature of the mutational event was examined in a set of 136 thioguanine resistant mutants selected from several tumorigenic and non-tumorigenic CHEF lines. Significantly different frequencies of point mutations were found compared with large partial or whole gene deletions in different cell lines. The clonal inheritance of specific mutational patterns as well as the high frequencies of large deletions were novel findings. Hypotheses to explain these results are discussed in relation to the known genomic instability of tumour cells. I am honoured by the opportunity to contribute to this collection of papers dedicated to Professor Guido Pontecorvo on the occasion of his eightieth birthday. For almost half of this time I have been privileged to enjoy his friendship, and to benefit from his influence at critical stages in my scientific development.     

Altered glycosylation of membrane glycoproteins in human uroepithelial cell lines

Total cellular glycopeptides of 7 human uroepithelial cell lines that differ in the grade of transformation (TGr) were analysed by gel filtration and affinity chromatography on immobilized lectins. The 4 cell lines that are tumorigenic in nude mice and invasive in vitro (TGr III) possess more highly branched, tri- and tetraantennary N-acetyllactosaminic glycans, with less biantennary glycans than the 2 non-tumorigenic, noninvasive (TGr II) cell lines examined. The only exception to this general pattern is the third cell line, which is classified as TGr II. The cellular glycopeptide distribution pattern in this cell line is similar to that of the TGr III cells. The possible relationship between altered glycosylation of membrane glycoproteins and the expression of a malignant phenotype is discussed.     

Mediated coalescence: a possible mechanism for tumor cellular heterogeneity

Recently, we demonstrated that tumorigenic cell lines and fresh tumor cells seeded in a 3D Matrigel model, first grow as clonal islands (primary aggregates), then coalesce through the formation and contraction of cellular cables. Non-tumorigenic cell lines and cells from normal tissue form clonal islands, but do not form cables or coalesce. Here we show that as little as 5% tumorigenic cells will actively mediate coalescence between primary aggregates of majority non-tumorigenic or non-cancerous cells, by forming cellular cables between them. We suggest that this newly discovered, specialized characteristic of tumorigenic cells may explain, at least in part, why tumors contain primarily non-tumorigenic cells.     

Gangliosides in human urothelial cell lines of different transformed phenotypes: the effect of v-raf-oncogene transfection

In a previous study we have shown that established human urothelial cell lines, representing grade of transformation II (TGr II, non-tumorigenic, non-invasive cells), are characterized by accumulation of the GM2 ganglioside as compared to cell lines of TGr III with tumorigenic and invasive properties. In the present study, the analysis of gangliosides from two tumorigenic sublines obtained after transfection of the TGr II cell line HCV 29 with the v-raf-oncogene, provided further evidence for the inverse relationship between tumorigenicity and the GM2 ganglioside expression. The two transfected sublines: T112C1 and T112D1, representing TGr III, were characterized by a decreased level of GM2 which was accompanied by an increased content of the GM3 ganglioside as compared to the parental HCV 29 cell line.     

The establishment and characterization of a new BALB/c angiosarcoma tumor system

A BALB-3T3/A31 untransformed cloned cell line and 3 selected variants derived from this parental cell line, expressing 3 increasingly malignant phenotypes, have been established and characterized in vivo and in culture. This new tumor series, identified as angiosarcoma, consists of an anchorage-independent non-tumorigenic cell clone and 2 sublines exhibiting tumorigenic and metastatic properties. Morphological examination revealed that the 3 transformed cell variants differed from the normal parental cells and were not contact-inhibited. Karyotype and rate of cell proliferation in culture were similar for all the cell variants. Cytoskeletal visualization by immunofluorescence staining revealed that the tumorigenic and metastatic cell lines expressed an altered organization of actin cables and a smaller number of vinculin-containing focal contacts. Lactoperoxidase iodination of cell surface proteins showed the appearance of an Mr 86,000 protein in the tumorigenic and metastatic cell variants. Analysis of cell-surface glycoproteins demonstrated an increased sialylation of Mr 66,000 and Mr 62,000 glycoproteins in the transformed, tumorigenic and metastatic cell lines. This angiosarcoma tumor model system allows investigation of cellular characteristics which might be relevant to specific stages in tumor progression.     

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.     

IGF-2 autocrine stimulation in tumorigenic clones of a human colon-carcinoma cell line

Clonal analysis has shown that the SW613-S human colon-carcinoma cell line is heterogeneous; some cell clones display a high level of amplification of the c-myc gene and are tumorigenic in nude mice, whereas others have a small number of copies of this gene and are non-tumorigenic. Tumorigenic clones can proliferate in a chemically defined serum-free medium, whereas non-tumorigenic clones cannot. Suramin, like anti-insulin-like growth factor (IGF) or anti-IGF-1 receptor antibodies, efficiently inhibits the growth of tumorigenic clones in defined medium. Inhibition by suramin or by anti-IGF antibodies can be reversed by pure IGF-1 or IGF-2. Pure IGF-1 or IGF-2 or culture medium conditioned by tumorigenic clones can stimulate DNA synthesis in cells of non-tumorigenic clones. Co-culture with cells of tumorigenic clones sustains the growth of non-tumorigenic clones in defined medium. Cells of both tumorigenic and non-tumorigenic clones express high-affinity IGF-1 receptors at their surface but tumorigenic clones produce on average 5 times more IGF-1 and 25 times more IGF-2 than non-tumorigenic ones. These results indicate that autocrine growth stimulation of tumorigenic clones by IGFs through the IGF-I receptor is essential for their ability to grow in defined medium. Since cells of tumorigenic clones produce IGF-2 at levels 80 times higher than IGF-1 and since an antibody strictly specific for IGF-1 has no effect on DNA synthesis in cells of tumorigenic clones grown in defined medium, IGF-2 is very likely the main effector in the autocrine loop. © 1995 Wiley-Liss, Inc.