Metabolically labeled cell membrane proteins in spontaneously and in SV40 virus transformed mouse fibroblasts.

A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.     

Angiogenesis-inducing ability of human bladder epithelium cell lines and "spontaneously" transformed murine fibroblasts

Ten human bladder epithelium cell lines were tested for their ability to induce blood vessel formation after intradermal injection into irradiated ST/a mice. Cell lines that were shown to be tumorigenic in nude mice, were able to evoke angiogenesis of a higher intensity than nontumorigenic cell lines. No difference was observed between the angiogeneic ability of tumorigenic cells originating from tumors and from in vitro transformed urothelium of nontumor origin. Similarly the origin of nontumorigenic urothelial cell lines did not show any influence on their angiogeneic abilities, but nontumorigenic cell lines which had undergone "infinite growth transformation" exhibited a higher angiogeneic activity than nontumorigenic cell lines with a finite life. The angiogeneic reaction evoked by human bladder epithelium cell lines showed cell dose- and time-dependence; but it was unrelated to the growth potential of the cultured cells. Two "spontaneously" altered sarcoma-producing murine cell lines showed a higher angiogeneic activity than tumorigenic human bladder epithelial cells. The angiogeneic response to these two murine cell lines was unrelated to morphological signs of transformation and to differences in growth rate, serum requirement, saturation density, anchorage dependence, and isoimmunizing properties.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

Genetic analysis of tumorigenesis. VIII. Suppression of SV40 transformation in cell hybrids and cytoplasmic transferants

Intraspecies somatic cell hybrids of BALB/c mouse 3T3 and SV40-transformed embryonic fibroblast (SVT2) cells were analyzed for transformation-associated properties and their tumorigenic potential in nude mice. In confirmation of our earlier findings, hybrids expressing the viral T-antigen were not suppressed for the ability to clone in medium with 1% serum. In contrast, division rate in medium with 1% or 10% serum, anchorage independence, cytochalasin-sensitive growth control, and tumorigenicity were suppressed noncoordinately, and the extent of suppression varied from one hybrid to another. Suppression was not simply determined by the increased chromosome content of the hybrid cells, nor was suppression correlated with rearrangements of the integrated viral sequence (SAGER et al., 1981a, b). Similar results were found in cytoplasmic transferants expressing T-antigen. Four independent transferants and subclones derived from them varied in the extent of suppression of anchorage independence and tumorigenicity. In both hybrids and transferants, a low serum requirement for clonal growth apparently was determined solely by expression of SV40 T-antigen, but other transformation properties, as well as tumorigenicity, appeared to require multiple changes in the cellular genome for their expression. These changes must occur during or after viral integration, since they are not expressed in uninfected 3T3 cells.     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.     

Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells.

Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells.     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.     

Anchorage-independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties

Calf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life-span of the lens cells and to inhibit the increase in size that occurs after 2-3 weeks of culturing the untransformed cells.     

Genetic analysis of tumorigenesis: I. Expression of tumor-forming ability in hamster hybrid cell lines

Two new fibroblasts cell lines from a male Chinese hamster embryo are described: one tumorigenic in nude mice (CHEF 16-2) and the other not (CHEF 18-1). Both lines have a stable diploid mode and mean of 22 with 10 pairs of homologous autosomes. The cell lines differ unambiguously in cloning ability in methylcellulose, colony morphology, and tumorigenicity; the expression of these traits was examined in a set of 18-1 X 16-2 hybrid clones. The results show initial suppression of tumorigenicity and anchorage independence, followed by growth in the nude mouse of subsets of cells with chromosomes reduced to the diploid range. Further studies are in progress to establish whether ability to grow in methylcellulose and in the nude mouse segregate coordinately at the cellular level, and whether this segregation has an identifiable chromosomal basis.     

Neoplasms produced from C3H/10T 1/2 cells attached to plastic plates; saturation density, anchorage dependence and serum requirement of in vitro lines correlated with growth aggressiveness in vivo

The C3H/10T 1/2 embryo cell line, which is nontumorigenic when inoculated subcutaneously in saline suspension, produces tumors when implanted subcutaneously attached to 1 X 5 X 10 mm plastic plates. Under these in vivo conditions there is direct selection for "spontaneous" transformants that have undergone the specific cellular alterations required for neoplastic behavior. This is in contrast to the conventional situation where transformants are obtained in vitro and are only secondarily tested in vivo for neoplastic behavior. Early passages of cell lines from four different C3H/10T 1/2 tumors explanted back in culture were quantitatively examined for tumorigenicity and for alteration in the properties of density inhibition, anchorage dependence, serum requirement, and plasminogen activator production. A fairly consistent quantitative relationship was found between the degree of growth aggressiveness in vivo and the degree of expression of these phenotypic markers of the transformed state in vitro during early passages of the cell lines after tumor explantation.     

Expression of transformation characters in colchicine-resistant tumor cells

Malignancy and anchorage independence of Djungarian hamster tumor cell lines resistant to different doses (0.1-5.0 micrograms/ml) of colchicine were studied. The clones with low colchicine resistance (15-20-fold) did not differ in tumorigenicity from parental cells. The TD50 for highly colchicine-resistant cells (200-800-fold) was several orders of magnitude higher than that for wild-type cells. Colchicine resistance did not affect the expression of the cells anchorage independence. The cloning efficiency in a semi-solid medium was the same both for colchicine-resistant cell lines and wild-type cells.     

Isolation of Defective Lysogens from Simian Virus 40-transformed Mouse Kidney Cultures

Rescue of simian virus 40 (SV40) from hamster and murine cell lines transformed by nonirradiated or by ultraviolet (UV)-irradiated SV40 (10(-3) to 10(-5) survival) was studied. A combination of tests was employed to detect induction of SV40 synthesis: (i) co-cultivation with susceptible monkey kidney (CV-1) cells; (ii) treating mixtures of transformed and CV-1 cells with UV-irradiated Sendai virus (UV-Sendai) prior to co-cultivation; and (iii) plating untreated or UV-Sendai-treated mixtures of transformed and CV-1 cells with freshly trypsinized CV-1 cells. The first and second tests provided a measure of the total infectious SV40 yield per culture, and the third test provided a measure of the frequency of induction (fraction of transformed cells giving rise to infectious centers). With the combination of tests, SV40 was rescued in all trials from TSV-5 hamster cells, mKS-BU100 mouse cells, and from several lines of mouse kidney cells transformed by UV-irradiated SV40 (mKS-U lines). The frequency of induction was about 7 x 10(-2) for TSV-5 cells, about 3 x 10(-3) for mKS-BU100 cells, greater than 10(-4) for the mKS-U lines which were "good" yielders, and about 10(-5) to 10(-4) for the mKS-U lines which were "average" yielders. SV40 of a plaque type different from parental virus was rescued from four of the mKS-U cell lines. Virus was also easily rescued from: (i) tumor cells produced from the mKS-A line of transformed mouse kidney cells; (ii) mouse kidney cells transformed by SV40 which had been rescued from mKS-BU100 cells; and (iii) tumor cells (HATS) which had been produced by inoculating newborn hamsters with SV40 rescued from mKS-BU100 cells. The frequency of induction of HATS cells was of the same order of magnitude as the frequency of induction of TSV-5 cells. In a study of the kinetics of virus induction, it was shown that SV40 could be detected 28, 40, and 48.5 hr after UV-Sendai treatment of mixtures of CV-1 and TSV-5, HATS, or mKS-BU100 cells, respectively. Although all of the mKS-U lines contained the SV40-specific tumor antigen, some were poor virus yielders (SV40 was recovered in less than 50% of the trials) and five lines were rare virus yielders (SV40 recovered only once in four or more trials). Forty-eight mKS-U lines were nonyielders; SV40 was never recovered by any test used thus far. UV-Sendai-treated mixtures of pairs of nonyielder mKS-U lines with CV-1 cells also did not yield infectious virus. Various factors affecting rescue have been discussed. The mKS-U lines which were poor virus yielders, rare yielders, or which never yielded virus have been classified tentatively as "defective lysogens" which contain mutational lesions at loci essential for detachment of SV40 from integration sites or for SV40 replication, or for both.     

Spontaneous neoplastic evolution of Swiss mouse embryo cells (PMS-14) in culture

Some biological properties related to transformation have been studied to characterize a spontaneously transformed mouse embryonic fibroblast cell line, PMS-14. Low population doubling time with moderately high plating efficiency and saturation density were noted. These cells showed reduced adhesion to substratum, enhanced agglutinability by concanavalin A, anchorage independent growth in soft agar and tumorigenicity in mice.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

"Transformation" of human endothelial cells by SV40 virions

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages. The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.     

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Characterization of chemically and virally transformed variants of Madin-Darby canine kidney (MDCK) epithelial cells

Oncogenic derivatives of Madin-Darby canine kidney (MDCK) cells were isolated in the nude mouse, and nononcogenic anchorage-independent transformants were isolated in vitro following chemical mutagenesis in vitro. These transformed cell lines as well as a Moloney sarcoma virus (MSV) transformed line were characterized with respect to their serum and anchorage requirements, growth rates, final saturation densities, and sensitivities to contact inhibition. None of these in vitro growth characteristics were found to correlate with tumorigenicity in nude mice. One tumorigenic clone, MDCK-T1, was characterized with respect to serum-free growth requirements, cAMP production, and ornithine decarboxylase (ODC) activity. These cells exhibited a significant reduction in the PGE1 requirement for growth, they produced higher levels of cAMP, and they expressed a reduced level of ODC activity relative to the parental MDCK cells. These findings may reflect changes in growth control mechanisms which accompany kidney epithelial cell tumorigenesis and suggest that the study of transformed lines derived in this manner could lead to the identification of in vitro properties which are associated with malignancy.