Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens.

The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation (C. Yasutake, Y. Kuratomi, M. Ono, S. Masumi, and M. Kuwano, Cancer Res. 47:4894-4899, 1987). Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.     

ENC1的克隆,原核表达与数种细胞系表达谱分析

以人 3月胎脑总RNA为模板 ,用RT PCR的方法得到了ENC1(ectoderm neuralcortex 1)基因的cDNA ,经测序证实该cDNA的长度为 180 0bp ,包含ENC1的完整编码区 .将之克隆入pGEX 4T 1载体构建重组表达质粒 ,转化大肠杆菌BL2 1表达 ,经Sepharose 4B纯化得到目的蛋白 .通过Northern印迹和RT PCR检验了该基因在数种细胞系中的表达 ,结果表明其在神经胶质母细胞瘤细胞系U2 5 1中有较高的表达 ,而在包括神经母细胞瘤细胞系SH SY5Y的其他数种细胞系中无表达 .与在正常生理状态下神经系统中两种细胞的表达情况相反 .这种分布不同的情况提示了ENC1在这两种不同来源的肿瘤的发生发展中具有不同作用     

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.     

Activation of a novel human transforming gene, ret, by DNA rearrangement

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.     

Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.     

Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas

DNAs from nitrosoethylurea-induced rat neuroblastomas transform NIH/3T3 mouse fibroblasts in a transfection assay. DNAs of such transformed cells can be used in a subsequent cycle of transfection to generate secondary foci that contain virtually no foreign genetic material besides the sequences carrying the rat neuroblastoma transforming function. These secondary neuroblastoma transfectants were injected into young mice and grew out into fibrosarcomas. Sera from these mice were examined for reactivity with any proteins which were induced specifically by the neuroblastoma transforming sequence. These sera precipitate a polypeptide of about 185,000 daltons from ³⁵S-methionine-labeled cell lysates of the rat neuroblastoma cells that served as DNA donors and in all transfection-derived primary and secondary foci. This protein is present in high levels in all neuroblastoma transfectant clones, but was not detectable in a variety of other transformed cells. Antisera were prepared from mice bearing tumors induced by transformed cells derived by transfection of DNAs from various tumor cell types unrelated to rat neuroblastoma. These antisera failed to immunoprecipitate the 185,000 dalton protein. These data indicate that the synthesis of the 185,000 dalton protein is specifically induced by the neuroblastoma transforming sequence. The protein may be encoded by the transforming sequence and may mediate transformation in this chemically induced tumor.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.     

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5''-fps sequence has a role in the transforming activity.

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.     

Analysis of cellular expression of gangliosides by gene transfection. II: Rat 3Y1 cells transformed with several DNAs containing oncogenes (fes, fps, ras & src) invariably express sialosylparagloboside

The transfection of several DNAs, containing oncogenes ras, src, fes or fps, into rat 3Y1 cells invariably induced the neosynthesis of sialosylparagloboside (SPG; IV3 alpha NeuAc nLcOse4Cer), with a concomitant decrease in GM3. All these oncogenes are 'extranuclear' type oncogenes, of which the products are expressed in the cytoplasm or on the cell surface membrane. These results are in striking contrast to those on the transfection of 'intranuclear' type oncogenes (ex., adeno E1) into the same 3Y1 cells, where GD3 neosynthesis was specifically brought about by the transfection.     

Human cell transformation in the study of sunlight-induced cancers in the skin of man

Human cell transformation provides a powerful approach to understanding--at the cellular and molecular levels--induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

Analysis of cellular expression of gangliosides by gene transfection. I: GD3 expression in myc-transfected and transformed 3Y1 correlates with anchorage-independent growth activity

Transfection of c-myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno El (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471-1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc-transformed 3Y1 cells, implying that GD3 plays some specific role in myc-induced transformation of 3Y1 cell line.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.