Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation

The radiobiology of human tumors suggests that multiple factors are involved in clinical radioresponsiveness. To date, no direct experimental evidence is available to correlate intrinsic cellular radiosensitivity with the steps of malignant transformation. We developed an in vitro multistage model of epithelial neoplasia using human epidermal keratinocytes to examine the effects of malignant transformation on radiation response. These cells were first immortalized as a result of infection with a hybrid virus (adenovirus 12 and simian virus 40) and subsequently transformed either by infection with a second virus (Kirsten murine sarcoma virus) or by treatment with a chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide). We demonstrate that primary human epidermal keratinocytes were radiation resistant (D0 = 2.24 Gy) as compared with human fibroblasts (D0 = 1.45 Gy) and that this resistance was retained in the immortalized as well as the transformed cell lines. These findings present direct experimental evidence that radiation sensitivity of malignant human keratinocytes is an intrinsic property of the precursor cell that may be conserved through the stages of neoplastic transformation.     

The study of multi-stage carcinogenesis in retinoblastoma and familial polyposis coli patient-derived skin fibroblast cell culture systems

Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.     

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Macromolecular changes accompanying immortalization and tumorigenic conversion in a human fibroblast model system

Mutagenesis of a diploid human fibroblast strain, KD, with the chemical carcinogen 4 nitroquinolin-1-oxide led to the isolation of stably immortalized neoplastic substrains. Four of these transformed strains, HuT-11, -12, -13, and -14, have been characterized in great detail with regard to morphology and changes in gene expression from the parental KD strain. The HuT-11, -12 and -13 substrains are immortalized and non-tumorigenic, in contrast to HuT-14 which is both immortalized and tumorigenic. The HuT-14 substrain expresses a defective beta-actin as a consequence of a point mutation in 1 of the 2 functional beta-actin alleles. All 4 HuT strains have induced expression of the phosphoprotein plastin and 2 EGF-related polypeptides, and down-regulated expression of the transformation-sensitive tropomyosin isoforms. KD and HuT cells expressing high levels of exogenous mutant beta-actin after gene transfection show morphological alterations. HuT-12 transfectants with excessive mutant beta-actin expression exhibit an elevated tumorigenic potential and tropomyosin-isoform switching characteristic of the tumorigenic HuT-14 strain.     

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

The subcellular localization of the p53 molecule was studied in transformed and non-transformed fibroblasts. A newly established transformed cell line obtained by treating primary embryonic mouse cells in vitro with the chemical carcinogen methylcholanthrene was compared with the embryonic parent fibroblasts. The transformed cells lost the spindle shape characteristic of the parent fibroblasts, acquired an accelerated growth rate, developed into tumors when injected into syngeneic mice and expressed high levels of p53 synthesis estimated by immunoprecipitation of [35S]methionine-labeled cell extracts. The cellular localization of the p53 molecule was studied by immunofluorescent staining of fixed cells with monoclonal antibodies and by immunoprecipitation of [35S]-methionine-labeled p53 from various subcellular fractions. p53 was mainly found in the nucleus of the transformed fibroblast, while in the parent non-transformed primary embryonic cells, p53 was detected in the cytoplasm in a Triton X-100 soluble fraction, and associated with the cytoskeleton. The modulated distribution of p53 was also confirmed by analyzing a wide range of independently established transformed and non-transformed fibroblastic cell lines growing in vitro. The switch from the cytoplasmic localization of p53 in the non-transformed fibroblasts to a chromatin-associated accumulation in the transformed cells suggests a possible mechanism by which this protein may function in the transformed fibroblasts.     

Transformation studies on human fibroblasts from familial polyposis coli patients and normal donors

Transformation experiments have been carried out on human diploid fibroblasts derived from normal individuals and those from 2 groups with dominantly inherited cancer predisposition, familial polyposis coli (FPC), and multiple endocrine neoplasia, type 2 (MEN-2). Treatment with a single or multiple doses of the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), resulted in limited anchorage-independent (AI) growth in both normal and FPC cultures; no permanent cell lines were produced but FPC cells showed increased proliferation with low doses of the carcinogen. Carcinogen treatment followed by application of the tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), for 38 weeks was insufficient to cause full transformation in cultures derived from normal people or MEN-2 patients although AI growth was induced in all 3 cell types. Three FPC cultures exhibited an extended life span over the solvent controls. Two of these are still actively dividing and have a clonal pseudodiploid karyotype.     

Intermediate filament expression and lifespan potential in human somatic cell hybrids

Summary Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids. This work was supported by grant no. AG 02664 from NIA (to C.L.B.) and by grant nos. 1R01 HD 18129-01 from NIH and PCM83-09068 from NSF (to R.H.S.). Editor’s Statement The tight correlation between the expression of the intermediate filaments of the immortal parent in hybrids of limited lifespan fibroblasts and HeLa cells with the transformed phenotype is of interest. It may offer important clues to the mechanism involved in cellular senescence. Gordon H. Sato     

Bovine endothelial cells transformed in vitro by benzo(a)pyrene

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid, Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid kayotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 10⁷ cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.     

Proline oxidase in cultured mammalian cells

We sought a cultured cell line with Proline Oxidase activity to study the regulation and physiologic role of the enzyme in mammalian tissues. Among the cell lines tested, only LLC-RK1 cells, derived from rabbit kidney, had significant Proline Oxidase activity; the Km for proline of the enzyme from these cells was similar to that for the liver enzyme. LLC cells, Proline Oxidase positive, were able to convert proline to CO2. In contrast, CHL cells, Proline Oxidase negative, did not have this capability. The presence of Proline Oxidase in LLC cells and the absence of the enzyme in fibroblasts suggest that Proline Oxidase may serve as a marker enzyme for distinguishing parenchymal kidney cells from fibroblasts in culture. Cells transformed by SV40 virus and cells transformed by methylcholanthrene had activities higher that the parent cell line, but this effect of transformation could not be generalized to all transformed cells. Finally, L-hydroxy proline at 100-fold greater concentration than substrate L-proline failed to decrease proline oxidation. This finding suggests distinct degradative enzymes for these two amino acids.     

Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

Biological properties of human melanoma cells in culture.

Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and that metastatic melanoma seems to display a higher degree of malignant transformation than the primary.     

Transformation of diploid human fibroblasts by transfection with the v-sis, PDGF2/c-sis, or T24 H-ras genes

Gene transfection techniques have provided powerful methods to examine the roles of cellular and retroviral oncogenes in the transformation process in rodent fibroblasts. However, the use of such techniques with diploid human fibroblasts has been limited. We have developed transfection procedures to reproducibly transfect such cells with oncogenes, and methods for the biological characterization of the transformants. We have shown that the v-sis and T24 H-ras oncogenes, as well as the platelet-derived growth factor gene (PDGF2/c-sis), are capable of inducing a transformed phenotype in normal diploid human fibroblasts, but are not capable of conferring infinite lifespan or making such cells tumorigenic.     

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.     

Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon

Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC alpha and PKC epsilon. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC alpha and a concomitant decrease in PKC epsilon, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2+ in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC beta 1, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the expression of PKC alpha and PKC epsilon are mediated mainly through a PKC-independent pathway. The present results provide the first evidence that transformation of cells by an oncogene can alter the relative expression of specific isoforms of PKC. It is possible that these changes contribute to the malignant phenotype of these cells.     

Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T-antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.     

Genes that cooperate with tumor promoters in transformation

Tumor-promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro-1 confers lifespan extension on these cells. Recently, we have found that a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced synthesis of two proteins of 15 and 16 kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation.     

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture

Primary monolayer cultures of normal and malignant human mammary epithelial cells were tested for fibronectin by indirect immunofluorescence using antisera specific for fibronectin. This protein was not detectable on either the normal or malignant epithelial cells. Similar results were obtained for normal and malignant mouse mammary epithelial cell cultures. Control normal and transformed fibroblasts exhibited the expected result: the normal cells were positive and the transformed cells were negative. With the use of supernatant fluids from the same cultures or an agar-overlay assay on viable cells, high levels of plasminogen-dependent fibrinolytic activity were detectable in both the normal and malignant mammary cells. Thus, two characteristics that distinguish normal from transformed fibroblasts do not serve as markers of malignancy in mammary epithelial/carcinoma systems.     

Immortalized cells as experimental models to study cancer

The development of cancer is a multi-step process in which normal cells sustain a series of genetic alterations that together program the malignant phenotype. Much of our knowledge of cancer biology results from the detailed study of specimens and cell lines derived from patient tumors. While these approaches continue to yield critical information regarding the identity, number, and types of alterations found in human tumors, further progress in understanding the molecular basis of malignant transformation depends upon the generation and use of increasingly sophisticated experimental models of cancer. Over the past several years, the recognition that telomeres and telomerase play essential roles in regulating cell lifespan now permits the development of new models of human cancer. Here we review recent progress in the use of immortalized human cells as a foundation for understanding the molecular basis of cancer.     

Characterization of Morphologic Revertants of Murine and Avian Sarcoma Virus-Transformed Cells

Morphologic revertants which contain avian or murine sarcoma viruses have previously been isolated at low frequency from clonal lines of transformed mammalian cells. In the present study, these lines have been further characterized. They are indistinguishable from nontransformed parent cell lines with respect to parameters such as saturation density and colony formation in depleted medium or on monolayers of contact-inhibited cells. The rate of glucose uptake had also reverted to normal. The malignant potential of one of the revertant lines was examined and found to be markedly reduced compared to that of the corresponding transformed cells. The differences in the susceptibilities of revertant cells to retransformation by the same or other oncogenic viruses suggest that different cellular genes may be involved in expression of transformation by various tumor viruses.     

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.