Inheritance of immunogenicity and metastatic potential in murine cell hybrids from the T-lymphoma ESb08 and normal spleen lymphocytes.

T-lymphoma cells were fused with normal lymphoid cells to examine the segregation of tumorigenicity and metastatic capacity in the hybrids. In independent fusions the immunogenic ESb08 T-lymphoma line fused successfully with normal syngeneic spleen cells (from DBA/2 and CD1 mice) enriched either with T-cells or B-cells. Ten times fewer hybrids were obtained with B-cells compared to the number obtained with T-cells, and marker assays showed that both types of fusions preferentially generated T-T hybridomas. Some of the hybrids resembled their tumor parent in their ability to form primary and secondary tumors only in irradiated DBA/2 mice, whereas other hybrids lost the high ESb08 immunogenicity, were equally tumorigenic, and in some cases metastatic, in nonirradiated mice. DNA distributions of the original hybrid lines ranged from a hexaploid DNA content (expected for complete hybrids derived from a tetraploid line and normal diploid cells) to a tetraploid DNA content, confirming the reported chromosome instability of T-T hybrids. No correlation was noted between the initial DNA content and tumorigenicity, but in the case of complete hybrids, reduction in the ploidy levels always was observed in the cells of primary and metastatic lesions. One chromosomally stable and highly malignant hybrid (C2), which was analyzed for segregation of chromosomes and for drug-resistance markers, showed preferential loss of chromosomes from the normal T-cell fusion partner. The decreased immunogenicity of this hybrid could not be related to any detectable loss of chromosomes from the ESb08 tumor parent.

Preservation of morphological, functional, and karyotypic traits during long-term culture and in vivo passage of two human skin squamous cell carcinomas.

Two cell lines (SCL-I and SCL-II) derived from squamous cell carcinomas of human skin were investigated during 4 years in culture. Both lines were tumorigenic in nude mice, and cells could be recultivated from xenografts. Growth in agar remained poor, but both cell lines developed abnormal stratified epithelial structures in organotypical cultures. The morphological and particularly ultrastructural characteristics remained typical in both cultures and xenografts. Keratinization slightly decreased, but nude mouse tumors differentiated as the original tumors, and this was reflected in keratin expression. Six major polypeptides (Mr 61,000, 57,000, 54,000, 51,000, 49,000, and 45,000) were similarly identified in both tumors and cell lines, also after animal passage, which was further substantiated by two-dimensional gel electrophoresis, but quantitative variations were found with different growth conditions. A distinct keratin cytoskeletal network was visualized in both lines by immunofluorescence, but only a few cells in SCL-II also expressed vimentin. Flow cytometry demonstrated 2c DNA stem lines for original tumors and derived lines. Early passages were hypodiploid by cytogenetic analysis of banded chromosomes. In SCL-I, a shift to tetraploidy occurred before passage 20 and remained stable throughout. In SCL-II, an incomplete shift to near tetraploidy and a stem line deviation were apparent, but later passages, nude mouse tumors, and cells recultured therefrom were hypodiploid (2c) again. Chromosome studies further revealed distinct stable marker chromosomes which showed additional structural aberrations with time in culture and after animal passage. Thus, phenotypically and genotypically, each squamous cell carcinoma and its derived cell line were distinct, and characteristics were preserved over long time periods in vitro and through in vivo passage.

Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.

The altered phenotypic expression and chromosomal characteristics of mouse and human malignant keratinocyte lines have been studied in vitro and in vivo (in comparison with normal primary cultures). The cell lines exhibited different morphologic aspects that are probably more related to their respective degree of differentiation than to different stages in malignancy. Although all cell lines studied were deficient in some aspects of keratinization, certain basic structural and biochemical features were maintained, and these may serve as valid criteria for the identification of their epithelial nature. The altered expression of keratin proteins and morphologic differentiation can be modulated under in vivo growth conditions, but they cannot be reverted toward normality. Chromosomal alterations (in number and structure) occur early and are highly indicative criteria for malignancy, even though no tumor-specific aberrations have been identified. Two new approaches for evaluating characteristics of abnormal growth and differentiation in vitro, and of invasiveness in vivo, have been developed and have proved sensitive test methods for identifying malignant cells. While several abnormalities in growth and differentiation of cell lines in vitro are highly indicative of their malignant nature, the final proof that they are tumor cells still requires in vivo assay. The transplantation assay for studying cellular invasiveness not only improves the sensitivity of in vivo malignancy tests but has also proved to be a valuable model system for elucidating the modulation of differentiation by external influences.

Spontaneous in vitro malignant transformation in a xeroderma pigmentosum fibroblast line.

This paper deals with a spontaneous malignant transformation in one of our XP fibroblast lines. This cell line, designated XP29MA, was derived from a 14-year-old boy who did not show skin tumors or precancerous alterations either at the time of clinical examination or when the biopsy was taken. We have compared the following features in both the malignant and the benign cell line from which the malignant line developed: tumor formation in nude mice, repair capacity, cytogenetic status, light and electron microscopic characteristics. The benign cell line XP29MA had a doubling time of 4.3 d, did not form tumors in nude mice, showed a very low repair capacity (as determined by colony-forming ability, unscheduled DNA synthesis and alkaline elution) but exhibited a normal cytogenetic and ultrastructural status. In contrast, the transformed cell line XP29MAmal grew three times faster, formed colonies in methyl cellulose, gave rise to fibrosarcomas in nude mice, showed a drastically higher repair capacity, and was characterized by an extreme genetic imbalance, resulting from numerical and structural chromosome alterations of Nos. 1, 3, 4, 8, 12, 16, 17, 18, 20 and 21. Ultrastructural examination revealed fusiform and polygonal cells, the latter exhibiting large indented nuclei, vesicular dilatations of the endoplasmatic reticulum and numerous lysosomes. The higher repair capacity in XP29MAmal cells is tentatively explained in terms of reversion, enhancement of post-replication repair and/or expression of SOS-type functions.

Phenotypic and genotypic differences between high- and low-metastatic related tumor lines and the problem of tumor progression and variant generation.

We have described a number of phenotypic and genotypic differences between defined tumor sublines from the Eb/ESb model system that differ greatly in metastatic capacity. Some of these differences are summarized in Figure 5. It can be seen that the differences are both structural and functional. They include differences in the glycosylation of membrane glycoproteins as well as differences in total membrane protein composition. The functional differences refer particularly to enzymatic activities, cell motility, and invasive capacity. In addition, a change in membrane fluidity and membrane dynamics was seen when we investigated membrane vesicles shed spontaneously from these tumor lines. ESb type cells were found to shed about three times as much membrane material as the low-metastatic, Eb cells. Since such vesicles carried degradative enzyme activity, they could play a role in the invasion process. The vesicles also carried TATA and thus could also play a role in preventing immunological attack against these tumor cells. Another immunological escape mechanism was found to be based on the ability of the high-metastatic cells to generate immunoresistant variants that no longer expressed the respective TATA. A cytogenetic comparison of such immunoresistant variants with the TATA+ line revealed several chromosomal changes. The contribution of chromosomal rearrangements to the ability of malignant cells to generate variants was discussed.

Cytogenetic changes during tumor progression towards invasion, metastasis and immune escape in the Eb/ESb model system.

Related tumor lines which represent different stages in their progression towards metastatic capacity were investigated and compared at the chromosomal level. The parental low-metastatic tumor line (L5178Y/Eb) was derived from a long-term transplanted, chemically induced T-cell lymphoma of the DBA/2 mouse. The cytogenetic analysis included this Eb line, a spontaneous high metastatic variant thereof which expressed a distinct tumor-associated transplantation antigen (ESb TATA+), and an immunoresistant TATA-negative variant of the latter (ESb TATA-). All three cell lines were characterized by a near-diploid chromosome count and by some common chromosomal markers derived from Nos. 6, 13 and 16 Large-scale chromosomal rearrangments resulted in the formation of eight marker chromosomes in Eb cells, 16 in ESb TATA+ cells and 18 in ESb TATA- cells. Tumor progression in this system showed a tendency to monosomies, which could bring the corresponding genes to a hemizygous state and possibly to a release from repression. Chromosome 15 was trisomic in Eb cells, monosomic in ESb TATA+ cells and hardly detectable in ESb TATA- cells. The Ig heavy-chain gene-carrying region of both chromosomes No. 12 was found in translocation with chromosomes Nos. 5, 13 and 14 (Eb cells) and with Nos. 1 and 17 (ESb cells). ESb TATA- cells differed from ESb TATA+ cells at four different chromosomes (Nos. 5, 8, 14 and 15).

Tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances sister chromatid exchanges and numerical and structural chromosome aberrations in primary mouse epidermal cell cultures.

The tumor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) moderately stimulated sister chromatid exchanges in primary epidermal cultures (PEC) from C3H mice, and strongly enhanced structural chromosome aberrations. In G-banded metaphases for TPA (10-(8) and 10-(6) M for 54 h) treated PEC aneuploidy (hypo- and hyperdiploidy) increased and structural aberrations were enhanced 8- to 10-fold. Breaks, fragments and metacentric chromosomes had raised 7- to 11-fold. Chromatid interchanges (tri- and quadri-radials) and centromeric splitting, virtually absent in controls, appeared in 4--8% of metaphases. The non-promoting 4-O-methyl-TPA did not induce chromosomal alterations. These substantial effects on the genetic material of target cells represent a new aspect of the mechanism of action of tumor-promoting phorbol esters.

Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin.

A human cell line (SCL-1) from a poorly differentiating cutaneous squamous cell carcinoma (SCC) ws studied through 20 passages during 2 years. Cells maintained their original morphology with low degree of keratinization, as indicated by light and electron microscopy. The keratin peptide pattern resembled the type in SCC tumors, and the corresponding filaments were detected by immunofluorescence at all passage levels. Cells did not grow in soft agar but formed tumor-like nodules in an "organotypic" culture assay (on lifted collagen gels) and grew invasively after transplantation to immunosuppressed inbred C3H mice. After injection into BALB/c nu/nu mice, tumors of SCC morphology were formed. The hypodiploid tumor stem-line was maintained for about 10 passages, when a shift to hyperploidy started, as determined by chromosome and DNA flow microfluorometric analyses. Two stable marker chromosomes (in 100 and 70% of the metaphases, respectively), involving chromosomes 7 and 9, strongly indicated a monoclonal origin of this cell line.