Antigenicity of clones of mouse prostate cells transformed in vitro

New cell-surface antigens were detected on nineteen of twenty-four malignant lines of 3-methylcholanthrene (MCA)- or MCA-epoxide-transformed cells derived from cloned C3H mouse prostate cells. Exhaustive cross-tests between lines derived from single control clones showed that in all but two lines these antigens were individually distinct and did not cross-react. Eight spontaneously transformed cell lines were also examined but only two of these were antigenic. The remaining six lines appeared to be non-immunogenic in syngeneic mice, although cell surface alloantigens were detectable. Non-transformed cells were not immunogenic in syngeneic hosts, whether or not they had been treated with MCA.     

c—myc基因对胃上皮细胞生物学特性影响的研究

目的探讨c—myc基因对于胃上皮细胞增殖状态、细胞周期、凋亡等生物学特性及浸润能力等表型的影响。方法将c—myc基因插入载体pcDNA3．1构建pc—myc真核表达载体。以脂质体介导转染HFE145正常胃上皮细胞建立稳定转染细胞系,而后使用流式细胞术、生长曲线法、平板克隆形成法、细胞迁徙法等方法分析稳定表达株相关生物学特性的变化。结果与转染pcDNA3．1空载体及空白HFE145细胞相比转染pc—myc载体的稳定表达细胞株生长显著加快,前5d细胞计数差异有统计学意义（P〈0．05）,后两组之间差异无统计学意义;细胞周期检测显示,pc—myc转染组处于G：～M期的细胞比例平均约25％,显著高于未处理的HFEl45细胞和转染pcDNA3．1空载体的细胞的5％（P〈0．05）,而处于s期的细胞比例平均约10％,显著均低于其他两组（P〈0．05）;流式细胞术检测结果显示,HFE—myc组细胞的凋亡比例平均5．8l％,显著高于两对照组（P〈0．05）;平板克隆形成实验结果显示,pc—myc转染组平均克隆形成率0．27,显著高于其他两组（P〈0．05）;细胞迁徙实验结果提示,pc—myc转染组穿膜率与其他两组相比差异无统计学意义（P〉0．05）。结论myc基因可能具有促进细胞生长增殖及分裂作用,同时可影响细胞周期,增加处于分裂期细胞的比例,可能具有促进细胞分裂作用,但对细胞迁徙能力的影响作用较小。     

The ability of normal mouse cells to reduce the malignant potential of transformed mouse bladder epithelial cells depends on their somatic origin

Somatic cell hybrids have been made between a transformed mouse bladder carcinoma cell line and normal mouse bladder epithelium and mesenchyme. In the epithelial tumour/mesenchyme hybrids the malignant phenotype was expressed dominantly whereas in the carcinoma/normal epithelium hybrids the malignant potential was greatly reduced. In both cases the dominant in vitro and in vivo phenotype was that of the normal parental cells. All hybrid tumours were first palpable after 4-7 days, demonstrating that the tumours had not arisen as a result of in vivo selection of a sub-population of tumorigenic cells. Chromosome analysis showed that the carcinoma/normal epithelium hybrids were all in the hypertetraploid range but the large variation in the karyotypic profile of each hybrid made it impossible to implicate any specific chromosomes in the control of expression of the malignant phenotype. During normal development in bladder epithelium, terminal differentiation is associated with tetraploid formation by cell fusion. The reduction in malignancy of the carcinoma/normal epithelium hybrids may perhaps be due to the expression of genes associated with normal terminal differentiation after cell fusion and tetraploid formation. This is also supported by the more differentiated phenotype of the hybrid tumours. Of the 10 mesenchyme/epithelium hybrids analysed cytogenetically , four were in the hypertetraploid range from which little meaningful data could be obtained about specific chromosome losses. Chromosome analysis of the cells from the near-tetraploid hybrids showed only minor differences from what might have been expected from the input of the two parents; these differences appeared to be due to random chromosome loss. The maximum number of chromosomes lost from any of the hybrids was five, although one, two or three was more usual. The only consistent chromosome loss was of a single copy of chromosome 4, which in two of the hybrids represented the only chromosome change. The possibility that this loss might facilitate re-expression of the malignant phenotype is discussed.     

Up-regulation of c-myc in a transformed cell line approaching stationary phase growth in culture

The present report describes a transformed cell line (AKR-MCA) in which the c-myc proto-oncogene is up-regulated by as much as 14-fold as cultures approach stationary phase growth. The untransformed counterpart AKR-2B cells did not exhibit such an increase in c-myc expression at high cell densities, nor did chemically transformed derivatives of another murine fibroblast cell line (C3H 10T1/2). N,N-Dimethylformamide and retinoic acid reduced c-myc levels in confluent AKR-MCA cells in association with a loss of transformed morphology, a reduction in saturation density, and the formation of a contact-inhibited monolayer at confluency. These findings suggest that the high levels of c-myc in confluent AKR-MCA cells may interfere with the normal signals involved in density-dependent growth regulation in this cell system. The effects of N,N-dimethylformamide and retinoic acid were reversible and dose-related. The half-time for the early, rapid decline in c-myc mRNA was approximately 26 min in response to N,N-dimethylformamide and 38 min in response to retinoic acid, effects which preceded the alterations in morphology and saturation densities. Activation of the latent transforming growth factor-beta in serum-free medium conditioned by confluent AKR-MCA cells, followed by its addition to preconfluent AKR-MCA cells, resulted in an up-regulation of c-myc mRNA. However, addition of serum-containing conditioned medium under similar conditions did not require prior acidification to up-regulate c-myc. Thus, active transforming growth factor-beta may be present in conditioned medium from confluent AKR-MCA cells grown in serum-containing medium, or autocrine factors other than TGF-beta may produce the confluency-associated up-regulation of c-myc and the altered density-dependent growth regulation in AKR-MCA cells.     

Antioxidant enzyme activities in normal and transformed mouse liver cells

Copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX, both Se-dependent and Se-independent), and glutathione reductase (GR) were measured in normal, nitrosoguanidine-transformed and SV40-transformed mouse liver cells in culture, as well as in mouse liver homogenates. Enzyme activities were compared on the basis of 3 different endpoints: per mg protein, per mg DNA, and per 10(6) cells. Except for GR, activity of all the measured anti-oxidant enzymes was much higher in vivo than in vitro. All of the anti-oxidant enzyme activities were lower in general in the 2 transformed cell lines than in the in vitro normal cell line, except Cu-ZnSOD, which showed little change. However, MnSOD was the only enzyme which showed lowered activity in both transformed cell lines, no matter what endpoint was used. This finding is in agreement with previous work showing lowered MnSOD activity in tumor cells.     

Molecular mechanisms of cell adhesion in normal and transformed cells

Summary Alterations in the adhesive mechanisms of cancer cells are likely to play an important role in determining the invasive or metastatic potential of these cells. An understanding of these alterations at the molecular level is now within reach, due to recent progress in the identification and characterization of several cell adhesion molecules (CAMs). Two of these molecules, the neural cell adhesion molecule N-CAM and the liver cell adhesion molecule L-CAM, are expressed on a variety of cell types from early embryos and throughout adult life, and appear to play several important roles in early inductive events, formation of specific intercellular connections, and maintenance of adult tissues. Two other molecules, the neuron-glia adhesion molecule Ng-CAM and a molecule involved in the specific adhesion of lymphocytes, appear to be more restricted in their developmental expression and function.The molecular characterization of N-CAM made possible for the first time an examination of the effects of transformation on the expression of a defined cell adhesion molecule. In both established cell lines from rat cerebellum and embryonic chick neuroepithelial cells, transformation by Rous sarcoma virus caused a large reduction in expression of N-CAM. In both cases, the N-CAM-mediated adhesion was correspondingly reduced. The neuroepithelial cells also became more highly motile after transformation. The decrease in N-CAM coupled with this increase in cell motility may significantly enhance the invasiveness of these cells. Other surface antigens have also been identified that may be involved in essential steps of invasion and metastasis.Such studies represent the initial step toward a detailed understanding of the role of CAMs in the various steps of metastasis. The accessibility of CAMs on tumor cell surfaces, and the availability of specific antibodies to these components suggests that reagents may become available in the near future that will offer new opportunities for preventing the formation of metastases.     

c-myc amplification coexistent with activating N-ras point mutation in the biphenotypic leukemic cell line RED-3

While activation of the protooncogene c-N-ras is observed regularly in acute myelogenous leukemia, amplification of c-myc in AML cells or derived lines is uncommon. In particular, concurrent ras/myc activation, which has been shown to be critical in several elegant models of malignancy, has been demonstrated in a very small number of human tumors or derivative cell lines. A cell line, RED-3, is described which was derived from cells of a patient with aggressive acute leukemia which exhibits many markers of lineage infidelity. DNA from this cell line contains an activating point mutation of c-N-ras as well as 20-30-fold amplification of c-myc. After HL-60, this is the second example of ras/myc activation in AML derived cells and demonstrates that this lesion is not unique to HL-60. Rather, it may be important in leukemogenesis in a small proportion of AML patients.     

Suppression of tumorigenicity of a human lung carcinoma line by nontumorigenic bronchial epithelial cells in somatic cell hybrids

Hybrid cell lines between HuT292-DM, a human lung carcinoma line resistant to 6-thioguanine and ouabain, and either normal human bronchial epithelial cells (NHBE) or an SV40 "immortalized" but nontumorigenic derivative thereof (BEAS-2B), have been isolated by double selection. Hybrids of NHBE and HuT292-DM cells senesced after 40-43 population doublings in culture. In contrast, hybrids of BEAS-2B and HuT292-DM showed no sign of a culture "crisis" and have an indefinite life span. HuT292-DM cells produced tumors in 100% of athymic nude mice with a mean latency of 27 days, whereas tumorigenicity was totally suppressed in 76% of the BEAS-2B x HuT292-DM hybrids, with a 2- to 3-fold increased tumor latency in the remaining 24% of these hybrids. While the hybrids are hypotriploid to hypotetraploid, the parental lines are hypodiploid. The growth of HuT292-DM cells is stimulated, whereas NHBE and BEAS-2B cells are inhibited by serum. The growth response of the BEAS-2B x HuT292-DM hybrids to serum is similar to that of HuT292-DM cells. Thus, tumorigenicity and culture longevity are dominantly controlled by the nontumorigenic parent (NHBE or BEAS-2B). On the other hand, serum responsiveness is more similar to that of the tumorigenic parent (HuT292-DM).     

Karyological patterns and expression of malignancy in some homologous mouse somatic hybrid cells

A homologous mouse somatic hybrid cell line (HyEN) was developed in mixed cultures of C3H mouse malignant cell line crossed with a BALB/c mouse non-tumorigenic cell line. An attempt was made to establish a possible relationship between the karyological features and die malignancy expression in this hybrid cell line as well as in six clones derived from it. As a rule, the karyotypes of the HyEN hybrid line and of its clones showed a marked dispersion of chromosome numbers and their modal values ranged from 80 to 129, the presumed “ideal” sum of the parental cell modes being 122. The HyEN line had a mode of 116 chromosomes and produced tumors when inoculated into histocompatible F₁mice. Among the six randomly isolated hybrid clones studied, the only non-tumor-producing one had a mode of 112–118, quite similar to those of two other malignant clones. Three more clones were malignant and had modes of 80, 98 and 129 chromosomes. Early cultures of cells recovered from these different tumors obtained were likewise submitted to karyological analysis which showed a tendency to a decrease in chromosomal mode values after passage in vivo. A similar though less important tendency was noted following in vitro passages of the original HyEN line as well as of its clones. However, in spite of the general tendency toward chromosomal loss, the hybrid cells participating in the formation of tumors represented a wide range of chromosomal variants and no conclusion could be reached concerning a definite relationship between chromosomal deletion and malignancy of the hybrid cells.     

Presence of two cytogenetic forms of amplified DNA: evidence for a role in tumor growth in an intraspecific mouse hybrid cell line

An intraspecific mouse hybrid epithelial cell line, F5/B, is described in which the homogeneously staining region (HSR)-containing marker chromosome from one parent is absent in about half of the cells. It is replaced in these cells by double minutes (DM), an alternative form of amplified DNA, which is liable to loss because of its instability at mitosis. DM probably arise from the breakdown of the HSR during clonal growth of F5/B. Subclones were derived possessing one or another cytogenetic feature, and their cloning efficiency in vitro and tumorigenicity in syngeneic animals were compared. There were no differences in in vitro tumorigenicity, but in vivo DM-containing subclones were significantly less tumorigenic than HSR-containing subclones or the F5/B parent hybrid. In tumors that developed after long latent periods, cells had increased numbers of DM compared with the inoculated population, demonstrating a selective advantage in vivo for cells with a high DM content. These results indicate a role for the amplified DNA in tumor growth.     

Heat shock protein synthesis and cell survival in clones of normal and simian virus 40-transformed mouse embryo cells

Exposure to hyperthermia induces the synthesis of a set of highly conserved polypeptides known as heat shock proteins (HSPs) in cells of most organisms. Since it has been suggested that these proteins may enhance cell survival by protecting cells from heat-inflicted damage, we studied the synthesis of the major HSPs (Mr 70,000 and 85,000) in clones of normal and SV40-transformed mouse embryo cells. These transformed cells had higher basal HSP levels and consistently synthesized the major HSPs at a higher rate both at physiological temperature and after exposure to heat shock (43-45 degrees). Parallel determination of cell survival showed that the transformed cells were, nevertheless, more susceptible to killing by hyperthermia than were their normal counterparts. Therefore, we conclude that the higher intrinsic resistance of the normal cells to killing by heat is not directly related to basal HSP levels or to the degree to which synthesis of these proteins is induced following exposure to hyperthermia. Considering the abnormal energy metabolism of transformed cells and the known sensitivity of HSP synthesis to energy source restriction, we hypothesize that both basal HSP levels and their induction by heat shock are related to alterations in energy metabolism.     

Growth characteristics in vitro of hybrids between normal and transformed cell lines

Sensitivity to the inhibition of division during cell crowding in vitro was determined for hybrids between normal and tumor cell populations of human and mouse origin. Interspecies hybrids were more sensitive to cell crowding than intraspecies hybrids. The results suggest that the inhibition of cell division due to crowding was more dependent upon the species of origin rather than on the normal or transformed character of the parental cell lines of the hybrid cell populations.     

Suppression of the cell proliferation and invasion phenotypes in glioma cells by the LGI1 gene

The leucine-rich, glioma-inactivated (LGI1) gene, located in 10q24, was originally identified because it was interrupted and inactivated by a reciprocal chromosome translocation in the T98G glioma cell line. Loss of LGI1 expression in high-grade brain tumors is correlated with the frequent loss of chromosome 10 during progression of gliomas. To investigate whether this gene can suppress the malignant phenotype in glioma cells, we introduced the LGI1 gene into cells that do (U87) and do not (T98G and A172) express LGI1 endogenously. A172 and T98G cells showed a significant reduction in cell proliferation potential as a result of re-expression of LGI1, whereas U87 cells did not. Using BD matrigel matrix chamber assays we were also able to show that the migration ability of the reconstituted A172 and T98G cells was also reduced considerably. Finally, these reconstituted T98G and A172 cells showed a significant reduction in the ability to form colonies in soft agar compared with the parental cells. This analysis clearly demonstrates that re-expression of the LGI1 gene in glioma cells that were null for its activity can greatly reduce their malignant potential. These observations provide the opportunity to investigate the role of LGI1 in gliomagenesis and, since LGI1 is predicted to be a membrane-bound protein, potentially provides the opportunity to develop novel treatment strategies for malignant gliomas.     

Alterations of mdm2 gene in x-ray transformed mouse 10t1/2 cell clones

Radiation-induced malignant transformation develops by a stepwise accumulation of molecular changes including mutation, amplification or overexpression of certain genes. Amplification and/or overexpression of mdm2 may be one of several molecular mechanisms for an altered growth control leading to the transformed phenotype. In the present investigation, we examined amplification, level of expression as well as mutation of mdm2 in radiation-transformed mouse C3H 10T1/2 cell clones. None of the clones examined showed structural changes of the mdm2 gene. However, mdm2 was amplified in 8 of 30 and overexpressed in 3 of 11 independent X-ray (600 cGy) transformed 10T1/2 cell clones, as compared with nontransformed, control or ultraviolet light (UVL)-transformed clones. None of the clones showing amplification and overexpression of mdm2 were among the 9 with alterations in the p53 gene. These results suggest that although amplification of the mdm2 gene may play a role in the transformation of some 10T1/2 cells, radiation-induced malignant transformation probably arises as a consequence of genetic events that involve several different pathways.     

Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line in culture

A method is described that permits the selection of spontaneously transformed mammary epithelial colonies from an untransformed mouse mammary epithelial cell line, NMuMG, and utilizes a long-term anchorage- independent growth of the transformants on soft agarose. These transformed cells (NMuMG-ST) are shown to be distinguishable from the untransformed cells by morphology, growth characteristics, induced carcinomas when transplanted into nude mice and ability to metastasize. This transformed phenotype displayed focal, multilayer growth and higher saturation density in comparison with the untransformed phenotype. Transplanted tumors as well as metastatic lung tumors in nude mice were adenocarcinomas morphologically similar to typical mammary tumors in humans. This selection procedure of mutant mammary cells from an immortalized cell line derived from normal mammary glands could be very useful to identify the genomic biomarkers in the growth regulation and malignant progression of breast cancer.