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1.
The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never
have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do
not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents.
All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole
chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing
the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated
in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic
kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MCF10A). These cell lines have abnormal
karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms
of cell immortalization by different “immortalizing agents”, oncogene-induced cell transformation of immortalized cells and
moderate response of the advanced tumors to anticancer therapy in the light of tumor “oncogene and chromosome addiction”,
intra-/intertumor heterogeneity, and chromosome instability. 相似文献
2.
Background
Nearly thirty years ago, it was first shown that malignant transformation with single oncogene necessarily requires the immortal state of the cell. From that time this thesis for the cells of human origin was not disproved. The basic point which we want to focus on by this short communication is the correct interpretation of the results obtained on the widely used human embryonic kidney 293 (HEK293) cells.Results
Intensive literature analysis revealed an increasing number of recent studies discovering new oncogenes with non-overlapping functions. Since the 1970s, dozens of oncogenes have been identified in human cancer. Cultured cell lines are often used as model systems in these experiments. In some investigations the results obtained on such cells are interpreted by the authors as a malignant transformation of normal animal or even normal human cells (as for example with HEK293 cells). However, when a cell line gains the ability to undergo continuous cell division, the cells are not normal any more, they are immortalized cells. Nevertheless, the authors consider these cells as normal human ones, what is basically incorrect. Moreover, it was early demonstrated that the widely used human embryonic kidney 293 (HEK293) cells have a relationship to neurons.Conclusions
Thus, the experiments with established cell lines reinforce the notion that immortality is an essential requirement for malignant transformation that cooperates with other oncogenic changes to program the neoplastic state and substances under such investigation should be interpreted as factors which do not malignantly transform normal cells alone, but possess the ability to enhance the tumorigenic potential of already immortalized cells. 相似文献3.
Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas. 总被引:6,自引:0,他引:6
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Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic. 相似文献
4.
E E Moore 《In vitro》1984,20(6):463-472
Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e.g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice. 相似文献
5.
Functional evidence for a second tumor suppressor gene on human chromosome 17. 总被引:4,自引:1,他引:3
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The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17. 相似文献
6.
Karyotypic variability has been investigated for nonimmortalized human embryonic lung cell line MRC-5, cultivated with Acholeplasma laidlawii strain PG-8 for 15-45 days. The character of cell distribution for chromosome number did not change during this time. In all investigated variants the number of polyploid cells increased considerably with the lengthening of the term after decryoconservation. The number of chromosomal aberrations in 15-45 days contaminated cells increased significantly as compared to the control at the expense of dicentrics (telomeric associations). The number of dicentrics had a tendency to increase with the lengthening of the term of contamination. Thus, in 45 days the number of dicentrics increased twice as much as that in 15 days. The increase of polyploids may be due presumably to the specific character of karyotypic variability in nonimmortalized cell lines with the long-term cultivation. Our present and previous results made it possible to suppose that the formation of dicentrics (telomeric associations) in nonimmortalized "markerless" cell line, following the long-term mycoplasmal contamination, may prove additionally the role played by dicentrics in cell adaptation to in vitro conditions whatever the degree of transformation may be--nonimmortalized line or immortalized nontumorogenic or high tumorogenic lines. 相似文献
7.
Emma E. Moore 《In vitro cellular & developmental biology. Plant》1984,20(6):463-472
Summary Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal
carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines
have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more
advanced cell lineages (e. g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements.
This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal
chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically
distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase
activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes.
The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and
desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice.
This work was supported in part by National Institutes of Health, Bethesda, MD, Grant CA-15823 and by a departmental gift
from R. J. Reynolds Industries, Inc. 相似文献
8.
Establishment of human embryonic stem cell lines is one the major achievements in the biological science in the XX century and has excited a wide scientific and social response as embryonic stem cells can be regarded in future as unlimited source of transplantation materials for the replacement cell therapy. To date human embryonic cell lines are obtained in more than 20 countries. In our country the embryonic stem cell researches are carried out in the Institute of Cytology RAS and the Institute of Gene Biology RAS. ESC lines are derived from placed in culture inner cell mass of human preimplantation blastocysts used in the in vitro fertilization procedure. Studies with human ESC go in several directions. Much attention is paid to the elaboration of the optimal conditions for ESC cultivation, mainly to the development of cultivation methods excluding animal feeder cells and other components of animal origin. Another direction is a scale analysis of gene expression specific for the embryonic state of the cells and corresponding signaling pathways. Many efforts are concentrated to find conditions for the directed differentiation of ESC into different tissue-specific cells. It has been shown that ESC are able to differentiate in vitro practically into any somatic cells. Some works are initiated to develop methods for the "therapeutic cloning", that is transfer and reactivation of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts. Of great importance is human ESC line standardization. However, the standard requirements for the cells projected for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines undergo genetic and epigenetic changes and, therefore, the cell line genetic stability should be periodically verified. The main aim of the review presented is a detailed consideration of the works analyzing the genetic stability of human and mouse ESC lines. Human ESC lines established in our and as well as in other countries couldn't be used so far in clinical practice. It is highly probable that undifferentiated ESC cannot be applied for therapeutic purposes because of the risk of their malignant transformation. Therefore, main efforts should be focused on the production of progenitor and highly differentiated cells suitable for transplantation derived from ESC. 相似文献
9.
Cell fusion techniques were used to derive mammalian host cell lines suitable for large-scale production of therapeutic proteins. Although the 293S cell line, of human embryonic kidney origin, is an excellent host cell for mammalian gene expression, these cells have a tendency to form large and tight aggregates in suspension cultures and bioreactors. To solve the problem of aggregation, 293S cells were fused to a human suspension cell line, 2B8 (a Burkitt's lymphoma derivative), using polyethylene glycol (PEG). The PEG-treated 293S and 2B8 cells were selected in a medium supplemented with hypoxanthine-aminopterin-thymidine and G418 (1 mg/ml) to eliminate nonfused cells. These hybrid clones, designated as HKB (hybrid of kidney and B cells), are negative for endogenous immunoglobulin expression. Most clones are readily adaptable to serum-free suspension culture under shaking conditions without forming large and tight aggregates. One clone, HKB11, was shown to support high-level expression of cytokines [interleukin (IL)-2 and IL-4], ICAM-1 and rFVIII in a side-by-side comparison with 293 and Chinese hamster ovary cells. The above-described characteristics of HKB cells indicate that HKB11 is a favorable cell host for the production of human therapeutic proteins. 相似文献
10.
Krelin Y Zhang L Kang TB Appel E Kovalenko A Wallach D 《Cell death and differentiation》2008,15(9):1350-1355
Caspase-8 is frequently deficient in several kinds of human tumors, suggesting that certain effects of this enzyme restrict tumor development. To examine the nature of the cellular function whose regulation by caspase-8 contributes to its antitumor effect, we assessed the impact of caspase-8 deficiency on cell transformation in vitro. Caspase-8-deficient mouse embryonic fibroblasts immortalized with the SV40 T antigen did not survive when cultured in soft agar, and were nontumorogenic in nude mice. However, the rate of transformation of these cells during their continuous growth in culture, as reflected in the observed emergence of cells that do grow in soft agar and are able to form tumors in nude mice, was far higher than that of cells expressing caspase-8. These findings indicate that caspase-8 deficiency can contribute to cancer development in a way that does not depend on the enzyme's participation in killing of the tumor cells by host immune cytotoxic mechanisms, or on its involvement in the cell-death process triggered upon detachment of the cells from their substrate, but rather concerns cell-autonomous mechanisms that affect the rate of cell transformation. 相似文献
11.
Chromosome 7 suppresses indefinite division of nontumorigenic immortalized human fibroblast cell lines KMST-6 and SUSM-1. 总被引:9,自引:3,他引:6
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T Ogata D Ayusawa M Namba E Takahashi M Oshimura M Oishi 《Molecular and cellular biology》1993,13(10):6036-6043
Using nontumorigenic immortalized human cell lines KMST-6 (KMST) and SUSM-1 (SUSM), we attempted to identify the chromosome that carries a putative senescence-related gene(s). These cell lines are the only ones that have been established independently from normal human diploid fibroblasts following in vitro mutagenesis. We first examined restriction fragment length polymorphisms on each chromosome of these immortalized cell lines and their parental cell lines and found specific chromosomal alterations common to these cell lines (a loss of heterozygosity in KMST and a deletion in SUSM) on the long arm of chromosome 7. In addition to these, we also found that introduction of chromosome 7 into these cell lines by means of microcell fusion resulted in the cessation of cell division, giving rise to cells resembling cells in senescence. Introduction of other chromosomes, such as chromosomes 1 and 11, on which losses of heterozygosity were also detected in one of the cell lines (KMST), to either KMST or SUSM cells or of chromosome 7 to several tumor-derived cell lines had no effect on their division potential. These results strongly suggest that a gene(s) affecting limited-division potential or senescence of normal human fibroblasts is located on chromosome 7, probably at the long arm of the chromosome, representing the first case in which a specific chromosome reverses the immortal phenotype of otherwise normal human cell lines. 相似文献
12.
Mei-Chih Liao Mihaela Diaconu Sebastian Monecke Patrick Collombat Charles Timaeus Tanja Kuhlmann Walter Paulus Claudia Trenkwalder Ralf Dressel Ahmed Mansouri 《World journal of stem cells》2014,6(2):248-255
AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice. 相似文献
13.
目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。 相似文献
14.
15.
Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines 总被引:3,自引:0,他引:3
M Oshimura H Kugoh M Koi M Shimizu H Yamada H Satoh J C Barrett 《Journal of cellular biochemistry》1990,42(3):135-142
The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors. 相似文献
16.
The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST-1i and STt-4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney-specific enzyme activity levels in 293, ST-1i, and STt-4i cells to determine their ability to exhibit kidney-specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (gamma-GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC-PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney-specific enzyme activities was assessed in response to several differentiation-inducing agents (adenosine, n-butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST-1i and STt-4i exhibit elevated levels of LAP, gamma-GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just gamma-GTP and maltase. Maltase and gamma-GTP enzyme activities in ST-1i and STt-4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST-1i and STt-4i are comparable to normal HEK cells in expression of kidney-specific enzymes and in responsiveness to differentiation-inducing agents, in spite of continued expression of SV40 oncogenes. 相似文献
17.
Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines 总被引:5,自引:0,他引:5
Gu Y Li H Miki J Kim KH Furusato B Sesterhenn IA Chu WS McLeod DG Srivastava S Ewing CM Isaacs WB Rhim JS 《Experimental cell research》2006,312(6):831-843
In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents. 相似文献
18.
We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types. 相似文献
19.
S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation. 相似文献
20.
The mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis 总被引:2,自引:0,他引:2
Gupta A Guerin-Peyrou TG Sharma GG Park C Agarwal M Ganju RK Pandita S Choi K Sukumar S Pandita RK Ludwig T Pandita TK 《Molecular and cellular biology》2008,28(1):397-409