Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells.

Our recent studies on the process of immortalization of cultured human mammary epithelial cells (HMEC) have uncovered a previously undescribed, apparently epigenetic step, termed conversion. When first isolated, clonally derived HMEC lines of indefinite lifespan showed little or no telomerase activity or ability to maintain growth in the presence of TGFbeta. Cell populations whose mean terminal restriction fragment length had declined to <3 kb also exhibited slow heterogeneous growth, and contained many non-proliferative cells. With continued passage, these conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good growth+/-TGFbeta, expression of high levels of telomerase activity, and stabilization of telomere length. We now show that exposure of the early passage conditionally immortal 184A1 HMEC line to the viral oncogenes human papillomavirus type 16 (HPV16)-E6, -E7, or SV40T, results in either immediate (E6) or rapid (E7; SV40T) conversion of these telomerase negative, TGFbeta sensitive conditionally immortal cells to the fully immortal phenotype. Unlike conditional immortal 184A1, the HPV16-E7 and SV40T exposed cells were able to maintain growth in TGFbeta prior to expression of high levels of telomerase activity. A mutated HPV16-E6 oncogene, unable to inactivate p53, was still capable of rapidly converting conditional immortal 184A1. Our studies provide further evidence that the transforming potential of these viral oncogenes may involve activities beyond their inactivation of p53 and pRB functions. These additional activities may greatly accelerate a step in HMEC immortal transformation, conversion, that would be rate-limiting in the absence of viral oncogene exposure.     

Establishment of human retinal pigment epithelial cell lines by oncogenes

The primary human retinal pigment epithelial cells were transfected with oncogenic sequences derived from viruses and cellular homologues of retroviral oncogenes 'protooncogenes' linked to simian virus 40 (SV-40) and retroviral promoters. Foci of cells were noted between 2 to 4 weeks after transfection. Individual colonies of cells were expanded from cultures transfected with SV-40 virion DNA, SV-40 large T antigen gene, Ha-ras oncogene, human and mouse c-myc and adenovirus E1A gene. Established cell lines tested were positive for the specific oncogene sequences by Southern hybridization and also expressed the protein as assayed by immunofluorescence and immunoblot analysis. Cell lines established with SV-40 large T antigen, and SV-40 virion DNA, exhibited epithelioid morphology up to the 25th passage and later became more rounded. However, all cell lines established with other oncogenes continued to retain epithelial morphology. Functional analysis of the cell lines demonstrated the presence of polarity and the ability to phagocytize rod outer segments, characteristics of retinal pigment epithelial cells. The use of oncogenes with immortalization/transformation potential may allow the establishment of cell lines from ocular tissues for analysing the biochemical basis of a disease like retinitis pigmentosa.     

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.     

Different types of DNA cleavage involved in osteosarcoma cell death

We investigated the effects of linoleic acid and eicosanoid synthesis inhibitors on the growth, DNA synthesis, and c-myc oncogene expression of human breast cancer cell lines. The cell growth of all cell lines examined was stimulated by linoleic acid, although the optimal concentration of linoleic acid for the growth stimulation differed among the cell lines. The growth stimulation of MCF7 and MDA-MB-435 cells was not dependent on the amount of released prostaglandin E(2) but their growth-concentration curve showed the same pattern as that of released leukotriene B-4. Moreover, linoleic acid stimulated the expression of c-myc mRNA in MCF7 cells without the presense of estrogen. Indomethacin as a cyclooxygenase inhibitor did not inhibit the c-myc expression in MCF7, but a lipoxygenase inhibitor, esculetin, markedly inhibited the c-myc expression in MCF7 affected by linoleic acid. These results suggest that linoleic acid has a direct mitogenic effect on breast cancer cells, and its mechanism is distinct from hormonal stimulation, Moreover, metabolites of lipoxygenase rather than those of cyclooxygenase may play an important role in the c-myc oncogene expression affected by linoleic acid.     

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.     

Loss of HPV-16 E7 dependence in cells transformed by HPV-16 E7 plus EJ-ras correlates with increased c-myc expression

We have previously shown that continued expression of the HPV-16 E7 gene was required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 plus EJ-ras. In this study we aimed to investigate the possible mechanisms by which cells could overcome this continued requirement for E7 expression. Using an inducible system for expression of the E7 gene we were able to generate cell lines no longer dependent on continued E7 expression. All such lines were still fully transformed, maintained inducible E7 expression and showed increased proliferative activity in the presence of HPV-16 E7 gene product. Further analysis has demonstrated a marked increase in the presence of c-myc protein in these cell lines when compared with the parental cells, suggesting a possible mechanism by which E7 dependence can be overcome.     

Effects of steroid hormones and peptide growth factors on protooncogene c-fos expression in human breast cancer cells

To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was mediated through activation of the c-fos protooncogene, we examined the expression of this oncogene in MCF-7 cells. In cells synchronized by double thymidine blockade, the peptide growth factors transforming growth factor alpha and epidermal growth factor increased c-fos mRNA levels 6-fold above controls after 30 min of treatment. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, increased c-fos mRNA levels 4- to 5-fold above control. 17 beta-Estradiol, a growth stimulator, increased c-fos mRNA levels less than 2-fold above control levels, while progesterone, vitamin D3, dihydrotestosterone, and dexamethasone had little effect on c-fos mRNA levels. In contrast, 17 beta-estradiol treatment initially diminished the c-myc RNA level after 30 min of treatment and resulted in an elevation of c-myc by 2.5 h after initiation of treatment. We conclude that c-fos induction in these cells is growth related and accompanies stimulation by transforming growth factor alpha and epidermal growth factor. 17 beta-Estradiol, on the other hand, induced much smaller increases in c-fos mRNA levels, suggesting an alternative or more complex mechanism of cellular stimulation.     

Alterations in growth properties of human papilloma virus type 16 immortalised human cervical keratinocyte cell line correlate with amplification and overexpression of c-myc oncogene

Clinical and epidemiological data support a role for human papilloma virus (HPV) type 16 in the pathogenesis of cervical carcinoma. The W12 cell line contains HPV16 sequences, predominantly as a high copy number episome, and is immortalised in vitro, but non-tumourigenic. A morphologically distinct sub-line of the parental W12 cell line was isolated which displayed increased growth rate, increased capacity for self-renewal and increased resistance to differentiation in comparison with the parental W12 cell line. The structure and expression of the HPV16 sequences was virtually identical in the two cell lines. However, expression of the c-myc proto-oncogene was elevated in the morphologically distinct sub-line, and this was associated with amplification, but not rearrangement, of the c-myc locus. Karyotype analysis demonstrated that amplification of the c-myc locus was a result of duplication of the long arm of chromosome eight.     

Oncogene transformation frequency of nonsenescent SFME cells is increased by c-myc

Immortalized, postcrisis mouse embryo cell cultures derived in serum-containing medium display genomic abnormalities and an altered, preneoplastic phenotype. These lines can be transformed with single oncogenes, such as Ha-ras, while efficient transformation of precrisis, genomically unaltered rodent embryo cultures require cooperating oncogenes, such as Ha-ras and the mouse c-myc gene constitutively expressed. Serum-free mouse embryo (SFME) cells, cultured under conditions in which serum is replaced by growth factors and other supplements, are 'immortalized' in the genomically unaltered state. SFME cells do not exhibit growth crisis or gross chromosomal aberration, and are dependent on epidermal growth factor for survival, growth inhibited by serum, and are nontumorigenic. Transformation of SFME cells can be achieved with ras alone, but the introduction of c-myc increased the transfection frequency upon subsequent transfection with ras by as much as twenty fold. Similar results were obtained with mutationally activated neu oncogene and with genomic human tumor DNA. Constitutive expression of c-myc alone did not alter the properties of the SFME cells. These results demonstrate that c-myc alters cellular responses to oncogenes in a culture system in which oncogene-induced immortalization is not a factor, indicating that the effects of myc may extend beyond an 'immortalization' function in these cells.     

Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA

A proliferating population of human foreskin keratinocytes (presently in the sixtieth passage) has been obtained after transfection with human papillomavirus (HPV) type 16 DNA. In contrast, the control cultures did not survive beyond the sixth passage. Cytogenetic analysis of cells taken from the twelfth passage revealed a heteroploid male karyotype. In approximately 50% of the cells a common marker chromosome was found, suggesting a clonal origin for at least part of the population. This is further substantiated by Southern blot analysis of cellular DNA which revealed oligomeric HPV 16 genomes integrated at a single site within the host DNA. RNA transcribed from the early region of the HPV 16 genome was identified in the cytoplasm. The immortalizing effect of HPV 16 DNA on human keratinocytes could be reproduced in a second experiment. Such cell lines represent an unique system to study the interaction of HPV with its natural target cell in vitro.     

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.     

Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry

A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.     

Effects of interferon-beta on Daudi cells and on small cell lung carcinoma cells which over-express the c-myc oncogene

The effects of human interferon-beta (IFN-beta) on four lung cancer cell lines, two of which over-express the c-myc oncogene, were compared to Daudi cells which also over-express c-myc. The anti-proliferative effect of IFN-beta on Daudi cells was correlated with a decrease in steady state levels of c-myc mRNA. IFN-beta treatment of a classic small cell lung carcinoma cell line and a non-small cell lung carcinoma cell line which express normal levels of c-myc did not decrease the rate of cell proliferation. IFN-beta, however, was found to have dramatic anti-proliferative effects on the two lung cancer cell lines with c-myc over-expression. The growth inhibitory effects of IFN-beta on the two lung cancer cell lines could not, however, be correlated with decreased amounts of c-myc mRNA. Furthermore, Western blot analysis showed that interferon treatment did not detectably affect the c-myc protein levels in these cells. Therefore, the mechanism by which IFN-beta slowed the growth rates of the two lung cancer cell lines did not appear to directly involve down-regulation of the c-myc oncogene, expressed in amplified amounts, in these cells.     

反义c-myc重组腺病毒的构建及其抗骨肉瘤细胞的作用

背景与目的:c-myc原癌基因在细胞的增殖调节中发挥重要作用,研究表明c-myc在骨肉瘤中常常扩增和过表达,而且具有促进细胞转化和诱导转移的特性。本文构建表达反义c-myc的重组腺病毒,并探讨其对不同p53基因型的骨肉瘤细胞系MG-63(P53缺失型)、U2OS(p53野生型)的影响。方法:应用基因重组技术构建表达反义c-myc的重组腺病毒(Ad-As-c-myc),并在体外转染MG-63、U2OS细胞,采用蛋白免疫印迹(Westernblot)、吖啶橙染色、RT-PCR、流式细胞仪(FCM)等方法检测或观察其对细胞c-mycmRNA表达、瘤细胞体外增殖、凋亡及细胞周期的影响。结果:成功构建Ad-As-c-myc,滴度可达2×109pfu/ml,体外转染MG-63、U2OS细胞后可明显抑制细胞的体外增殖,而且对携带野生型p53的U2OS更敏感。Ad-As-c-myc转染48h后可降低c-mycmRNA表达。吖啶橙染色及FCM检测证实,转染Ad-As-c-myc可诱导骨肉瘤细胞凋亡,细胞周期分析显示转染Ad-As-c-myc的MG-63细胞出现G2/M期阻滞,而U2OS细胞出现G1期阻滞。结论:腺病毒介导反义的c-myc能以p53依赖或非依赖性途径诱导骨肉瘤细胞凋亡,并抑制骨肉瘤细胞的增殖。