Tumor Dormancy: Death and Resurrection of Cancer As Seen through Transgenic Mouse Models

Cancer is caused by genetic changes that activate oncogenes or inactivate tumor suppressor genes. The repair or inactivation of mutant genes may be effective in the treatment of cancer. Drugs that target oncogenes have shown to be effective in the treatment of some cancers. However, it is still unclear why the inactivation of a single cancer associated gene would ever result in the elimination of tumor cells. In experimental transgenic mouse models the consequences of oncogene inactivation depend upon the genetic and cellular context. In some cases, oncogene inactivation results in the elimination of all or almost all tumor cells through apoptosis or terminal differentiation. However, in other cases, oncogene inactivation results in the apparent loss of the neoplastic properties of tumor cells, that now appear and behave like normal cells, however, upon oncogene reactivation rapidly recover their neoplastic phenotype. These observations illustrate that oncogene inactivation can result in a state of tumor dormancy. Understanding when and how oncogene inactivation induces sustained tumor regression will be important towards the development of successful therapeutic strategies for cancer.     

Oncogene expression in human hepatoma cells PLC/PRF/5

The expression of 7 cellular oncogenes in a human hepatoma cell line PLC/PRF/5 was studied using Northern blot analyses. Among the oncogenes tested, c-abl, c-fes, c-fms, c-myc, c-Ha-ras and c-sis were expressed. The oncogene c-Ki-ras was not expressed. The length of the mRNAs expressed was almost consistent with published data. Compared to the oncogene expression in Daudi lymphoma cells, the same kind of oncogenes were expressed in PLC/PRF/5 cells, but the intensity of the signal in each oncogene expression was stronger in Daudi cells than in PLC/PRF/5 cells. Considering the cellular localization and the function of each oncogene, the oncogene survey in hepatoma cells broadens the knowledge of hepatocarcinogenesis and the character of human hepatoma cells.     

反义核酸在肿瘤研究中的应用

反义核酸研究已活跃于肿瘤研究及基因治疗领域,反义核酸通过碱基配对待异性地抑制基因表达,因此为研究肿瘤中癌基因和生长因子的功能及癌基因突变检测提供了更为有效的手段,并为肿瘤的基因治疗提供了可能途径.文章综述了反义核酸在基因治疗中所面临的问题及部分解决办法.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses.

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.     

Phospholipase C-gamma 1 directly associates with the p70 trk oncogene product through its src homology domains.

Tyrosine phosphorylation of proteins was examined in NIH3T3 cells transformed by an oncogenic form of the trk protein. Proteins of 148, 140, 70, and 55 kDa were phosphorylated on tyrosine residues in trk-transformed cells but not control NIH3T3 cells. The 70-kDa protein may represent the trk oncogene protein itself which was shown to be tyrosine-phosphorylated in vivo using trk-specific antiserum. Phospholipase C-gamma 1 (PLC-gamma 1) was also found to be constitutively tyrosine-phosphorylated in trk-transformed cells and the trk protein co-immunoprecipitated with PLC-gamma. The GTPase-activating protein of ras (GAP) and the 62-kDa GAP-associated protein were tyrosine-phosphorylated in trk-transformed cells, and a lesser amount of trk co-immunoprecipitated with GAP relative to with PLC-gamma. The trk oncogene product bound specifically to a bacterially expressed fusion protein containing the src homology domains of PLC-gamma. The data suggest a significant role for PLC-gamma in intracellular signaling by the trk oncogene.     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

表皮生长因子对neu基因表达的诱导作用（简报）

The erb B2/neu oncogene encodes a protein which sequence is closely similar to the epidermal growth factor receptor (EGFR). We have previously found that EGF can induce the expression of erb B1/EGFR gene in normal and 3H-TdR transformed C3H/10T1/2CL8 mouse embryo fibroblast cells i.e. NC3H10 and TC 3H10 respectively, but we do not know whether the neu oncogene expression can be induced by EGF. In this study, the effect of EGF on NC3H10 and TC3H10 has been observed by Northern blot analysis. The result indicated that EGF had a obvious induction effect on neu oncogene expression in these cells. Thus, the expression of both erbB 1/EGFR gene and erbB 2/neu oncogene can be induced by EGF. This result may provide a novel clue to the molecular mechanism of EGF action in cell nucleus.     

表皮生长因子对neu基因表达的诱导作用(简报)

Shih等首次通过NIH/3T3细胞转染试验在乙基亚硝脲(ENU)诱导的大鼠神经胶质纤维瘤中分离鉴定出一种转化基因,称之为neu基因,其表达可导致培养的NIH/3T3细     

Recessive (mediator-) revertants from c-H-ras oncogene-transformed NIH 3T3 cells: tumorigenicity in nude mice and transient anchorage and serum independence of the recovered tumor cells in culture.

We have reported earlier the isolation of two recessive, serum- and anchorage-dependent revertants (R116 and R260) from a c-H-ras oncogene-transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity. We report here that neither revertant could be re-transformed by the K-ras or N-ras oncogene (though they could be re-transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double-minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage-independent growth.     

Mutagenic effect of the SV40 oncogene: induction of resistance to 6-mercaptopurine and serum independence

The mutagenic and transforming activity of SV40 DNA fragment, corresponding to its oncogene (the gene for large T antigen) was studied in Chinese hamster cells. After expression time of 3 to 4 days, the oncogene induced mutations of resistance to 6-mercaptopurine (6MP), while the DNA encoding the SV40 late genes, as well as DNA of Chinese hamster cells, were devoid of mutagenic activity. The value of induction ranged from 10(-4) to 10(-5). After the same expression time, the oncogene induced a typical character of oncogenic transformation - independence of serum growth factors (ser+). The value of induction of ser+ variants was somewhat higher than for resistance mutations. The study of 12 clones induced by the oncogene has shown the ser+ character to be hereditary, the expression of viral oncogene being not necessary for its maintenance. The data obtained support the hypothesis in favour of the participation of mutations of cellular genes in viral carcinogenesis.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.     

Transformation of NIH/3T3 to Anchorage Independence by H-Ras Is Accompanied by Loss of Suppressor Activity

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.