Malignant Transformation Of Mammalian Cells Research Articles

Caspase-3 promotes oncogene-induced malignant transformation via EndoG-dependent Src-STAT3 phosphorylation

Accumulating evidence suggests that caspase-3 plays critical roles beyond apoptosis, serving pro-survival functions in malignant transformation and tumorigenesis. However, the mechanism of non-apoptotic action of caspase-3 in oncogenic transformation remains unclear. In the present study, we show that caspase-3 is consistently activated in malignant transformation induced by exogenous expression of oncogenic cocktail (c-Myc, p53DD, Oct-4, and H-Ras) in vitro as well as in the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse model of breast cancer. Genetic ablation of caspase-3 significantly attenuated oncogene-induced transformation of mammalian cells and delayed breast cancer progression in MMTV-PyMT transgenic mice. Mechanistically, active caspase-3 triggers the translocation of endonuclease G (EndoG) from mitochondria, which migrates to the nucleus, thereby induces phosphorylation of Src-STAT3 signaling pathway to facilitate oncogenic transformation. Taken together, our data suggest that caspase-3 plays pivotal role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells.

ATM Paradoxically Promotes Oncogenic Transformation via Transcriptional Reprogramming

The role of the ataxia-telangiectasia-mutated (ATM) gene in human malignancies, especially in solid tumors, remains poorly understood. In the present study, we explored the involvement of ATM in transforming primary human cells into cancer stem cells. We show that ATM plays an unexpected role in facilitating oncogene-induced malignant transformation through transcriptional reprogramming. Exogenous expression of an oncogene cocktail induced a significant amount of DNA double-strand breaks in human fibroblasts that caused persistent activation of ATM, which in turn enabled global transcriptional reprogramming through chromatin relaxation, allowing oncogenic transcription factors to access chromatin. Consistently, deficiencies in ATM significantly attenuated oncogene-induced transformation of human cells. In addition, ATM inhibition significantly reduced tumorigenesis in a mouse model of mammary cancer. ATM and cellular DNA damage response therefore play a previously unknown role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells. SIGNIFICANCE: These findings uncover a novel pro-oncogenic role for ATM and show that contrary to established theory, ATM does not always function as a tumor suppressor; its function is however dependent on cell type.

The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

Tissue-specificity of proteoglycans expression in different cancers

Background. Proteoglycans (PGs) are complex glycosylated molecules playing an important role in cell-cell and cell-matrix interactions and signaling. Expression of PGs and their expression pattern change considerably during malignant transformation of mammalian cells and tissues. Objective. The aim of our work was to investigate tissue-specificity of main PGs expression (glypican-1, perlecan, syndecan-1, aggrecan, versican, CSPG4/NG2, brevican, decorin, lumican) in normal cells (fibroblasts and normal epithelial prostate cells PNT2) and in different human cancer cell lines (prostate, breast, lung, brain, kidney). Expression patterns of main PGs were determined in these cells using reverse transcription polymerase chain reaction analysis and immunocytochemical staining. Results. It was shown that fibroblasts actively expressed PGs, and PNT2 cells had lower (5–6-fold) expression levels of a limited set of PG. In different cancer cell lines, overall transcriptional activities of PGs varied up to 10-fold, although their expression patterns had tissue-specific properties (for example, expression of syndecan-1 is more specific for prostate cancer cells, while perlecan is typical for lung cancer cell lines). Conclusions. Along with this, variability of the PG expression patterns in cell lines of the same tissue of origin was shown, suggesting a possible contribution of the variable PGs expression to intratumoural heterogeneity of cancer cells and their potential as perspective biomarker (s) for personalised cancer diagnostics.

How a replication origin and matrix attachment region accelerate gene amplification under replication stress in mammalian cells.

The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

PAI-1 Expression Is Required for HDACi-Induced Proliferative Arrest inras-Transformed Renal Epithelial Cells

Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed renal cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was previously found to be dependent on transcriptional activation of the PAI-1 (SERPINE1) gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators). NaB-initiated PAI-1 expression preceded induced cell spreading and entry into G1 arrest. To assess the relevance of PAI-1 induction to growth arrest in this cell system more critically, two complementary approaches were used. The addition of a stable, long half-life, recombinant PAI-1 mutant to PAI-1-deficient v-K-ras-/c-Ha-ras-transformants or to PAI-1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of PAI-1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. The transfection of ras-transformed cells with the Rc/CMVPAI expression construct, moreover, significantly elevated constitutive PAI-1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level PAI-1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure.

Overexpression of ATA1/SLC38A1 Predicts Future Recurrence and Death in Chinese Patients with Hilar Cholangiocarcinoma

System A amino acid transporter is a Na+-dependent active transport system, mediating the uptake of amino acids, dysregulation of which has been found to be associated with malignant transformation in mammalian cells. However, the role of ATA1 in hilar cholangiocarcinoma is unclear. Here, we investigated ATA1 expression and determined its clinical significance in hilar cholangiocarcinoma. Tissue microarray blocks containing tumor specimens obtained from 48 patients were constructed. Expression of ATA1 in these specimens was analyzed using immunohistochemical studies. ATA1 overexpression was observed in 22 cases (44.9%). Overexpression of ATA1 was significantly associated with lymph node metastases. ATA1 expression has a significant correlation with recurrence and poor survival in univariate analyses. Multivariate analyses revealed that ATA1 was an independent predictor for future recurrence in patients with cholangiocarcinoma. Increased expression of ATA1 is frequent in human hilar cholangiocarcinoma and significantly correlated with the progression of cholangiocarcinoma, suggesting the importance of ATA1 in cancer development and progression. ATA1 expression may be used to predict recurrence and death and can serve as a promising target for therapy of this malignancy.

Gene Expression Changes and Induction of Cell Proliferation by Chronic Exposure to Arsenic of Mouse Testicular Leydig Cells

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Chronic exposure of arsenic has been shown to induce malignant transformation of mammalian cells; however, the mechanism underlying arsenic-induced carcinogenesis is not clear. The (1) induction in the cell proliferation, (2) decrease in DNA repair capacity resulting in the accumulation of mutations, and (3) changes in the DNA methylation patterns affecting regulation of genes are hallmarks of cancer development. Thus, the purpose of this study was to determine whether long-term exposure of both low and high concentrations of arsenic can perturb cell proliferation, DNA repair, and the maintenance of DNA methylation status in TM3 cells, an immortalized Leydig cell derived from normal mouse testis. The effect of arsenic on cell proliferation was determined by cell count data, and arsenic-induced gene expression changes were measured by quantitative real-time polymerase chain reduction (PCR). The results this study revealed a concentration-dependent induction of cell proliferation by arsenic. Increased expression of cell proliferation marker genes (PCNA, CyclinD1) and DNA methylation (DNA Methyl Transferase I) and decreased expression of genes for DNA repair (DNA Polymerase β, ERCC6) with lower concentrations of arsenic was also observed. Thus, the findings of this study are novel, as they indicate a mechanism for arsenic-induced cancers. This is based on the observed increase in cell proliferation and decrease in the capacity of cells to maintain its genomic stability. Our study provides the evidence that arsenic may play a role in the etiology of testicular cancer.

Genetic and epigenetic changes induced by chronic low dose exposure to arsenic of mouse testicular Leydig cells

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Genotoxicity of arsenic has been a topic of controversy. Both genetic alterations (mutations) and epigenetic changes (methylation) have been shown to play a crucial role in environmental carcinogenesis. Chronic exposure to arsenic has been shown to induce malignant transformation of mammalian cells. However, the genetic aberrations induced by arsenic in this process are unclear. The purpose of this study was to determine if both lower (1 pg/ml) and higher concentrations (100 ng/ml) of arsenic induces either mutations or methylation changes that could lead to the development of genomic instability in TM3 cells, immortalized Leydig cells derived from normal mouse testis. Two independent exposure times were used in this study which resulted in cells of 33 and 100 generations in age. Arsenic-induced genetic and epigenetic changes were screened at a genome-wide level by random amplified polymorphic DNA (RAPD), also known as AP-PCR method with undigested DNA as well as DNA digested by the methylation sensitive isosizomeric restriction enzymes MSPI and HpaII and untreated controls. Changes in the DNA fingerprint of both, the restriction enzyme digested DNA (indicating methylation changes) as well as undigested DNA (indicating mutations) from arsenic-treated (low as well as high dose) samples were observed as compared to their controls. Thus, this study provides the first evidence at DNA sequence level for mutagenic potential of arsenic. Further characterization of these altered genomic regions is underway. The understanding of these genetic and epigenetic changes in arsenic-induced carcinogenesis will provide a basis for better interventional approaches in both the treatment and prevention of arsenic-induced cancer.

Induction of Constitutive High-Level Expression of c-Myc in 32D Cells by Mycoplasmas is Associated with Their Ability to Prevent Apoptosis and Induce Malignant Transformation

Our previous studies showed that infections by mycoplasmas could prevent apoptosis and induce the malignant transformation of mammalian cells. Other studies indicate that c-Myc plays an important role in promoting apoptosis and malignant transformation of cells. To understand the role of c-Myc in the mycoplasma-induced apoptosis prevention and malignant cell transformation, 32D cells, an IL-3 dependent cell line, were infected and transformed by different species of mycoplasmas. The expression of Myc and ras gene families, apoptosis and the cell cycle during the infection and transformation were examined. Results showed that c-Myc expression was significantly increased in mycoplasma-transformed 32D cells. Withdrawal of IL-3 substantially decreased c-Myc expression in non-infected 32D cells and led to cell cycle arrest at the G1 phase followed by rapid apoptosis. Infection by M. fermentans or M. penetrans not only alleviated the sharp decrease of c-Myc expression, rescued 32D cells from cell-cycle arrest and prevented apoptosis in IL-3-free culture, but also induced autonomous growth of 32D cells. Although M. hominis and M. salivarium had the ability neither to prevent apoptosis nor to induce malignant transformation, they were still able to rescue the cells from cell cycle arrest at the G1 phase. The expression of ras family did not change significantly during the infection and transformation. These results suggest that constitutive expression of c-Myc appears to be associated with the continuous growth and malignant transformation of 32D cells induced by M. fermentans and M. penetrans, but not with rescuing the cell cycle arrest by the mycoplasmas.

Malignant Transformation of Mammalian Cells Initiated by Constitutive Expression of thePolo-like Kinase1

Polo-like kinase (Plk) is the mammalian homologue of the Drosophila polo and Saccharomyces cerevisiae CDC5 genes, which are thought to be involved in regulating chromosomal segregation. Previously, we showed that transient ectopic expression of Plk could induce DNA synthesis in quiescent NIH 3T3 cells, suggesting that Plk might also have a function during G1 or S phase. Here we report that microinjection of Plk mRNA is sufficient to drive quiescent cells into mitosis and that constitutive expression of Plk in NIH 3T3 cells causes oncogenic focus formation. These transformed cells grow in soft agar and form tumors in nude mice. Because Plk expression has been shown to be high in various human tumors, we suggest that Plk may contribute to the promotion and/or progression of human cancers.

The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.

Zebra fish myc family and max genes: differential expression and oncogenic activity throughout vertebrate evolution.

To gain insight into the role of Myc family oncoproteins and their associated protein Max in vertebrate growth and development, we sought to identify homologs in the zebra fish (Brachydanio rerio). A combination of a polymerase chain reaction-based cloning strategy and low-stringency hybridization screening allowed for the isolation of zebra fish c-, N-, and L-myc and max genes; subsequent structural characterization showed a high degree of conservation in regions that encode motifs of known functional significance. On the functional level, zebra fish Max, like its mammalian counterpart, served to suppress the transformation activity of mouse c-Myc in rat embryo fibroblasts. In addition, the zebra fish c-myc gene proved capable of cooperating with an activated H-ras to effect the malignant transformation of mammalian cells, albeit with diminished potency compared with mouse c-myc. With respect to their roles in normal developing tissues, the differential temporal and spatial patterns of steady-state mRNA expression observed for each zebra fish myc family member suggest unique functions for L-myc in early embryogenesis, for N-myc in establishment and growth of early organ systems, and for c-myc in increasingly differentiated tissues. Furthermore, significant alterations in the steady-state expression of zebra fish myc family genes concomitant with relatively constant max expression support the emerging model of regulation of Myc function in cellular growth and differentiation.

Application of Transmission Electron Microscope and Scanning Electron Microscope in experimental tumor studies

Transmission electron microscope(TEM) and scanning electron microscope(SEM) were widely used in experimental tumor studies. They are useful for evaluation of cellular transformation in vitro, classification of histological types of tumors and treating effect of tumors. We have obtained some results as follows:1. Studies on the malignant transformation of mammalian cells in vitro. Syrian golden hamster embryo cells(SGHEC) were transformed in vitro by ThO2 and/or ore dust. In a few days after dust added into medium, some dust crystals were phagocytized. Two weeks later, malignant transformation took place. These cells were of different size, nuclear pleomorphism, numerous ribosomes, increasing of microvilli on cell surface with various length and thickness, and blebs and ruffles(Figs. 1,2). Myelomonocytic leukemic transformation of mouse embryo cells(MEC) was induced in vitro by 3H-TdR. Transformed cells were become round from fusiform. The number of mitochondria and endoplasmic reticulum was reduced, ribosomes and nucleoli increased, shape of nuclei irregular, microvilli increased, and blebs and ruffles appeared(Fig. 3).

Translocation of chromosomes 16 and 18 in oxygen radical-transformed human lung fibroblasts

Human lung fibroblasts (MRC5) were treated chronically with the oxygen radical-generating system hypoxanthine plus xanthine oxidase and assayed for malignant transformation in the soft agar colony assay. After a thrice weekly exposure to oxygen radicals for 4 and 5 weeks, there was a significant number of transformants compared to controls. In 4 separate experiments, karyotypes of the malignant transformants were examined. 22 75 metaphases exhibited karyotypic abnormalities and in 13 22 of the abnormal karyotypes, a t16:18 (p13.3,q21) translocation was observed. This genetic lesion may represent a marker for oxygen radical-induced malignant transformation in mammalian cells.

Malignant transformation of mammalian cells in culture, including human cells.

This overview of the malignant transformation of mammalian cells in culture, including human cells, describes the earliest evidence of spontaneous, virus-induced, and carcinogen-induced transformation. It discusses several systems developed to assay the carcinogen-induced transformation of highly selected infinite life span ("established") cell lines as well as finite life span diploid cells. Evidence is presented to support the multistep hypothesis of the process of malignant transformation, and the theoretical requirement for acquisition of an infinite, or greatly extended, life span in culture if a cell is to become malignant is explained in light of the multistep nature of the process. The use of oncogene transfection studies to analyze the number and kinds of changes involved is discussed, with emphasis on studies using human cells. Finally, the results of earlier studies on viral- and carcinogen-induced transformation of mammalian cells (or chicken cells) are reinterpreted in the light of more recent insights into the process of carcinogenesis.

The mutagenicity and carcinogenicity of hair dyes

Synopsis A number of the constituents of semi-permanent and permanent hair dyes have been shown to be mutagenic in bacteria, to induce mutation, chromosome breakage, sister chromatid exchanges and malignant transformation in mammalian cells, to cause mutation in Drosophila, to induce mitotic recombination in yeast and to induce tumours in rodents. Some hair dye ingredients and commercial dyes have been shown to give rise, in the urine of treated rats, to metabolites which are also mutagenic in bacteria. The test systems used are briefly described and the data generated are reviewed. In addition, attempts to identify whether the hazards predicted from the laboratory studies present a risk to man either by epidemiological studies or monitoring of exposed populations are also discussed.

Induction of immortality is an early event in malignant transformation of mammalian cells by carcinogens.

Limited lifespan in normal diploid mammalian cells in culture is well documented1–3 and there is evidence that it is also a property of normal tissues serially transplanted in vivo4,5. In contrast, in favourable growth conditions, cells derived from tumours or premalignant tissue often exhibit unlimited proliferative potential (immortality) both in vitro and in vivo2,4,6–10. Malignant transformation of diploid cells in culture by carcinogens has been clearly demonstrated in several laboratories2,11–13. However, although it has been frequently reported that establishment in culture is one of many alterations in cell behaviour associated with transformation, the induction of immortality and its role in malignancy have not previously been subjected to serious scientific examination. Here, we show that treatment of Syrian hamster cell cultures with carcinogenic chemicals can induce rare immortal variants, the progeny of which then frequently progress to anchorage independence and malignancy after a further period of growth. In addition, by studying the properties of cell lines that are either immortal or anchorage-independent but not both, we have obtained evidence that immortality, although insufficient by itself, may be a prerequisite for malignant transformation.

Role of elevated temperature in malignant transformation of mammalian cells in vitro.

Rat embryo cells subjected in vitro to transient incubation at an elevated temperature (39 degrees C) became transformed and induced fibrosarcomas in both homologous and heterologous hosts. Malignant transformation correlated with the occurrence of karyotypic changes which appeared long after incubation at 39 degrees C and subsequent return to 37 degrees C. Control cultures incubated at only 37 degrees C did not show similar chromosomal changes or induce tumors and remained predominantly diploid during the same observation period. In contrast to rat embryo cells, marmoset monkey cell cultures incubated at 39 degrees C did not develop characteristics of transformed cells.