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.     

Differential gene expression in tumorigenic and nontumorigenic HeLa x normal human fibroblast hybrid cells

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable hybrids that are nontumorigenic and no longer express the HeLa tumor-associated marker intestinal alkaline phosphatase (IAP). Previous studies of spontaneous tumorigenic segregants from the nontumorigenic hybrids implicated the loss of one copy of human fibroblast chromosome 11 in the concomitant reexpression of tumorigenicity. In an attempt to identify genes involved in the control of tumorigenic expression, we performed differential display screening of nontumorigenic hybrid cells and tumorigenic segregants. Subsequent northern blot analyses reproducibly showed 17 differentially expressed genes, eight of which were expressed differentially in the nontumorigenic hybrids and nine of which were expressed differentially in the tumorigenic hybrids. The former were genes for 80K-L protein (a substrate of protein kinase C), AXL/UFO (a receptor tyrosine kinase), insulin-like growth factor binding protein 3, apolipoprotein AI regulatory protein, collagen type I alpha-2 chain, transforming growth factor-beta-induced gene product 3 (BIGH3), pregnancy-specific beta-1-glycoprotein, and fibroblast activation protein alpha. The latter nine genes were genes for serum/glucocorticoid-regulated kinase (SGK; a serine/threonine protein kinase), PTPCAAX1 (a tyrosine phosphatase), CXCR-4 (a G-protein-coupled membrane receptor), L-kynurenine hydrolase, beta-1, 4-galactosyltransferase, keratin 8, keratin 17, and H19 and a novel gene. The differential expression of these genes provided several interesting candidates for regulation of tumorigenic expression, including those involved in signal transduction and the extracellular matrix, cytoskeletal proteins, cell-surface enzyme, and the H19 gene.     

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.     

Isolation of alkylating agent-sensitive Chinese hamster ovary cell lines

Six derivatives of a Chinese hamster ovary cell line have beenisolated by selection for hypersensitivity to the cytotoxiceffects of the monofunctional alkylating agent methyl methanesulphonate (MMS). These cells are up to 6-fold more sensitiveto MMS than the parental line, as estimated from D₃₇ values,and are cross-sensitive to methyl nitrosourea, as well as tothe ethyl derivatives of these drugs. Comparisons of their sensitivitiesto the bifunctional alkylating agents cis-platinum (II) diamminedichloride, mitomycin C and melphalan reveals marked phenotypicdiversity, with only one mutant, designated MMS-2, exhibitingappreciable hypersensitivity to all of these agents. No strikinghypersensitivity to radiation or to the purine analogue caffeineis apparent in any of the mutant lines. Based on their profilesof sensitivity to DNA damaging agents, it would appear thatthese mutants are phenotypically unlike any previously describedmammalian cell mutants and probably represent a number of differentgenetic complementation groups. These mutants may facilitatean investigation into the mechanisms of repair of alkylationdamage in mammalian cells and could prove to be suitable hostsfor the cloning of human DNA repair genes.     

Two spontaneously transformed cell lines derived from the same hamster embryo culture

From a single primary culture of pooled hamster embryos, two morphologically distinct, malignant cell lines have evolved in the absence of experimentally applied selective factors, chemical, physical or viral. The cells of both sublines are male. One subline, designated Nil-1, is extremely pleomorphic with lobulated nuclei and micronuclei. It was tumorigenic at the 35th passage (18 weeks) after initiation of the primary culture. The second subline, designated Nil-2, is fibroblastic and required 30–45 more subcultures than Nil-1 to become tumorigenic. The malignant properties of Nil-1 and Nil-2 were not correlated with any specific chromosome changes. At a time when Nil-2 cells were still sensitive to contact inhibition and were not tumorigenic, they could be transformed by polyoma virus but not by SV40. The parent primary culture, however, was susceptible to transformation by SV40. Neither subline contained SV5 or lymphocytic choriomeningitis viral antigen or specific complement fixing antigen induced by polyoma virus, SV40, Rous sarcoma virus or adenovirus types 7, 12, 18, 21, or 31. No cytopathogenic agent and no mycoplasma could be isolated from the parent culture or sublires; no new transplantation antigen could be demonstrated. The possibilities in the etiology of the transformation are discussed.     

Secretion of proteinase inhibitors by tumorigenic and nontumorigenic guinea pig and Syrian hamster fibroblasts: evidence for autocrine regulation of local proteolysis

A cytokine that inhibits fibrinolysis has been detected in the serum-free culture medium of guinea pig and hamster fibroblasts. This proteinase inhibitor was also present in Triton X-100 extracts of guinea pig cells. It was stable at pH 3.0 for 2 hr and was produced by cells rather than assimilated from serum in the culture medium as evidenced by: (a) an apparent molecular size (less than 45 kilodaltons) less than that of the principal serum-derived proteinase inhibitors; (b) its continued secretion after several passages of the cells in serum-free medium; and (c) the lack of inhibitory activity in the medium of mitomycin C-treated cells. The cytokine inhibited the proteinase activity of human urokinase, soluble TPA-stimulated guinea pig plasminogen activator, and the cell-associated plasminogen activator of tumorigenic guinea pig cells. Soluble plasminogen activator appeared to be inhibited to a greater degree than the cell-associated enzyme. The fluorogenic substrate (7-(N-carbobenzoxyglycylglycylargininamido)-4-methylcoumarin was used in a direct assay of proteinase activity and demonstrated that the cytokine inhibited both plasminogen activator and plasmin, the two proteinases of the fibrinolytic cascade. Tumorigenic guinea pig and hamster fibroblasts as well as nontransformed guinea pig fibroblasts were found to produce the inhibitory cytokine, and the amount of inhibitor secreted was independent of the tumorigenic potential of the cells. Production of the inhibitor by normal cells may be related to contact inhibition of growth, and this cytokine may contribute to the fine regulation of local proteolysis within tissues.     

Spontaneous, malignant transformation of hamster embryo cells in vitro

Primary hamster-embryo cells were dispersed in vitro. The frequency of colony development depended on the original method used for dispersing the cells and on the number of cells seeded. Cell populations derived from passages in vitro of 3 colony isolates produced tumors when inoculated into baby or adult hamsters. Similar populations derived from primary or confluent, passaged cell cultures failed to produce tumors. The transformed cells were long, parallel Fibroblasts or short, randomly arranged spindle types. It is suggested that malignant, spontaneous transformation of cells occurs within a limited number of passages in vitro and that these cells may be a factor in virus-induced cell transformations.     

Highly elevated ultraviolet-induced mutation frequency in isolated Chinese hamster cell lines defective in nucleotide excision repair and mismatch repair proteins

We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair. By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins. Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity. Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.     

Topoisomerase II-dependent and -independent mechanisms of etoposide resistance in Chinese hamster cell lines

Resistance to etoposide (VP-16), amsacrine (mAMSA), and doxorubicin (Adriamycin) was studied in two Chinese hamster cell lines primarily selected for resistance to the epipodophyllotoxin. Both lines demonstrated profound resistance to VP-16, and mAMSA stimulated DNA breakage. However, the resistance to mAMSA cytotoxicity in both lines was less than expected from the level of resistance to the effects of topoisomerase II inhibition. Similarly, resistance to the cytotoxicity of high VP-16 concentrations in one of the lines was less than expected from the resistance to inhibition of topoisomerase II. An analysis of the relation of DNA breaks to drug cytotoxicity suggests that cross-resistance to mAMSA was mainly conferred through loss of mAMSA-stimulated, topoisomerase II-mediated DNA breaks. This mechanism also contributed towards reduced VP-16 cytotoxicity. However, our studies suggest that additional mechanisms, independent of resistance to VP-16-mediated topoisomerase II effects, greatly increased the resistance to this agent. Resistance to VP-16 cytotoxicity, not dependent on resistance to drug-mediated DNA cleavage, could be overcome at high drug concentrations in one of the resistant lines and might be responsible for the greater relative resistance to VP-16 than to mAMSA. These findings suggest the presence of two distinct mechanisms of resistance to VP-16 cytotoxicity, one presumably mediated by topoisomerase II and dependent on resistance to drug-mediated DNA scission, and a second mechanism independent of the effects of the drug on topoisomerase II.     

Growth and transplantability of clonally related tumorigenic murine cell lines.

A malignant cell line derived from the s.c. inoculation of Adenovirus 12 into a CBA mouse has been isolated in vitro, cloned, and within 10 passages the clones have been investigated for their karyotype, morphology, growth rate, saturation density and response to plant lectin in vitro, and their tumorigenicity and growth rate in vivo. The cell lines rapidly acquired a highly heterogeneous karyotype, but remained homogeneous with respect to more complex physiological parameters. Examination of the cellular characteristics has indicated that the rate of growth of the cell lines in vivo, but not their tumorigenicity, may be related to their in vitro potentials. The clones responded differently to the cytotoxic effects of concanavalin A, but there was no correlation between the effect of the lectin and the malignant potential of the cells.     

A conserved region in human and Chinese hamster X chromosomes can induce cellular senescence of nickel-transformed Chinese hamster cell lines.

Cellular senescence is the genetically programmed cessation of cellular proliferation. We have recently mapped a putative senescence gene(s) on the X chromosome of Chinese hamster embryo (CHE) cells. In the present study, we have utilized microcell-mediated chromosome transfer (microcell fusion) to test whether: (i) the human X chromosome exhibits similar genetic potential to induce senescence and (ii) the deletion or inactivation of the X-linked senescence gene(s) in CHE cells is associated with nickel-induced immortalization. A normal CHE or human X chromosome was first introduced into mouse-cell hybrids, then transferred by microcell fusion into a nickel-transformed, immortal male CHE cell line (Ni-2/TGR) with an X deletion (Xq1). Microcell fusion of the normal CHE X chromosome into tumorigenic Ni-2/TGR cells yielded senescence of all X recipient clones. The normal human X chromosome induced dominant senescence of tumorigenic Ni-2/TGR cells in only 17% of the resulting microcell hybrids (14/81). Karyotypic analyses of 13 non-senescing human X chromosome-derived microcell hybrid clones revealed that none of these clones retained the complete X. A normal CHE X chromosome induced senescence of 75% of hybrids obtained with another immortal and tumorigenic nickel-transformed male CHE cell line (Ni-6/TGR), which exhibited no visible deletion of the X chromosome, while the normal human X chromosome, only induced senescence in 19% of these hybrids. Transfer of the normal CHE or human X chromosome into spontaneously transformed and tumorigenic cell lines, CHO/TGR or V79/TGR, had little or no effect on their growth. These data suggest that both human and CHE cells possess similar X-linked genetic activities that regulate the process of cellular senescence, and that in Chinese hamster cells nickel-induced immortalization but not that of CHO or V79 cells is associated with inactivation of an X-linked senescence gene.     

纳米银对中国仓鼠肺成纤维细胞的细胞毒性和遗传毒性

目的： 研究纳米银对中国仓鼠肺成纤维细胞(CHL)的细胞毒性和遗传毒性并探讨其作用机制。方法：以梯度浓度(2.5、5.0、10.0、20.0、40.0和80.0 μg/mL)纳米银染毒CHL细胞,MTT比色法计算IC50以评估纳米银的细胞毒性水平。利用PI染色分析细胞周期变化并计算细胞增殖指数;Annexin V-FITC和PI双染检测细胞凋亡和坏死发生率,同时观察细胞染毒后的形态变化,记录前向角散射(FSC)和侧向角散射(SSC)信号;Giemsa染色中期分裂相涂片分析染色体畸变发生率。结果：纳米银毒性水平与纳米银浓度呈正相关(r=0.804,P<0.05),IC50为21.68 μg/mL。22.0 μg/mL纳米银与CHL细胞接触24 h后,细胞形态发生明显改变;细胞群SSC信号显著增强,为细胞摄入纳米银提供间接证据;细胞周期在G2/M期被捕获,对G0/G1期、S期以及细胞增殖指数变化情况影响显著(P<0.01);22.0 μg/mL纳米银染毒CHL 24 h后凋亡发生率与对照组差异明显(P<0.01);各浓度纳米银诱导染色体结构畸变率均大于5%(P<0.01),中高浓度四倍体发生率达到溶剂对照组的10倍以上(P<0.01)。结论：纳米银对CHL细胞有显著的细胞毒性和遗传毒性,线粒体功能受损、细胞凋亡在纳米银的细胞毒性和遗传毒性中起到重要作用。     

Cholesterol sulfate accumulation in tumorigenic and nontumorigenic rat esophageal epithelial cells: evidence for defective differentiation control in tumorigenic cells

In this study the regulation of squamous cell differentiation in several rat esophageal epithelial cell lines is examined. Nontumorigenic RE-149 cells undergo a program of squamous cell differentiation at confluence. This program of differentiation is influenced by the concentration of calcium in the medium and by the presence of retinoic acid. High calcium concentration stimulates terminal cell division, as indicated by a reduction in colony-forming efficiency, and increases the expression of the differentiated phenotype as indicated by an increase in cholesterol sulfate accumulation and cross-linked envelope formation. Retinoic acid inhibits squamous cell differentiation as both cholesterol sulfate accumulation and cross-linked envelope formation are reduced. Two tumorigenic cell lines, RE-B2 and RE-2BT, do not undergo squamous cell differentiation in vitro. High calcium concentration in the medium did not significantly reduce colony-forming efficiency or induce cross-linked envelope formation. High calcium concentration or retinoic acid had only a limited effect on the accumulation of cholesterol sulfate. RE-B2T cells exhibit high levels of cholesterol sulfate and cholesterol sulfotransferase activity. These levels appear no longer controlled by calcium or retinoic acid, indicating that the synthesis of cholesterol sulfate occurs in a constitutive manner. The altered responses of RE-2B and B2T cells to calcium and retinoic acid suggest that these malignant cells have acquired one or more defects in the control of differentiation.     

Spontaneous immortalization rate of cultured Chinese hamster cells

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.     

Identity of tumorigenic human urothelial cell lines and 'spontaneously' transformed sublines.

Restriction fragment length polymorphism (RFLP) analysis, comparative marker chromosome analysis, and polymorphic enzyme analysis was carried out on a total of eight human urothelial cell lines and sublines selected according to our knowledge of their HLA-A,B phenotype. RFLP analysis and cytogenetic analysis showed that the cell lines Hu1703He, Hu1922, and T24 are genuine cell lines of different origin. The identity of Hu1703He could not be confirmed by its isozyme phenotype which was identical to the T24 phenotype. RFLP analysis and isozyme analysis revealed that three cell lines, Hu456, Hu549, and Hu961a, and two transformed sublines, HCV-29Tmv and Hu609Tmv, are sublines of T24. A common origin of Hu456, Hu549, Hu961a, HCV-29Tmv, and Hu609Tmv was confirmed by marker chromosome analysis. However, the T24 origin of these cytogenetically related cell lines was not supported by chromosome analysis of T24. RFLP analysis and HLA phenotyping of two tumorigenic and invasive sublines isolated from a culture of non-tumorigenic Hu609 cells showed that non-tumorigenic Hu609 cells can transform ''spontaneously'' in vitro into tumorigenic Hu609T cells. The results emphasise the need for careful monitoring and screening of cell lines for their identity using more than one identification parameter.     

Photosensitizing dyes for the selection of nontumorigenic revertants from human lung cancer cell lines

Certain positively charged, lipophilic dyes have been noted by various authors to localize selectively in the mitochondria of carcinoma cells. Oseroff et al. (Proc. Natl. Acad. Sci. USA, 83:9729-9733, 1986) studied 10 carcinoma-specific mitochondrial photosensitizers and judged N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC) to be the most effective in selective carcinoma cell photolysis, a system where light-absorbing molecules accumulate only in carcinoma cells and on illumination initiate a reaction that kills or damages those cells. The present study duplicated the published EDKC retention result for the normal monkey kidney epithelial cell line CV-1. A series of nontumorigenic and tumorigenic human bronchial epithelial and human pleural mesothelial cells were assayed for EDKC uptake and retention, with the intent of using selective carcinoma cell photolysis to isolate nontumorigenic revertants of the tumorigenic lung cell lines. In addition, the uptake and retention of the fluorescent, mitochondria- and carcinoma-specific dye rhodamine-123 were surveyed in a series of hybrids between tumorigenic and nontumorigenic human bronchial epithelial cells. The half-life of dye retention ranged from 6 to 12 h in all the bronchial epithelial and mesothelial cells studied, with little or no dye selectivity for tumorigenic cells. When EDKC-retaining bronchial epithelial cells were illuminated with red light, significant reductions in short term viability and colony-forming efficiency were seen, which became more pronounced as light and dye doses were increased. However, these effects did not correlate with tumorigenicity within the cell series. The method, therefore, does not appear generally useful for the selection of nontumorigenic variants of human bronchial epithelial or pleural mesothelial cancers of the lung.