Malignant Transformation Of Human Fibroblasts Research Articles

Aneuploidy-inducing gene knockdowns overlap with cancer mutations and identify Orp3 as a B-cell lymphoma suppressor.

Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.

Retraction: Sp100 as a Potent Tumor Suppressor: Accelerated Senescence and Rapid Malignant Transformation of Human Fibroblasts through Modulation of an Embryonic Stem Cell Program

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.

Sp100 as a Potent Tumor Suppressor: Accelerated Senescence and Rapid Malignant Transformation of Human Fibroblasts through Modulation of an Embryonic Stem Cell Program

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts

BackgroundThe activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain.MethodsUnder the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays.ResultsExpression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions.Conclusion(s)The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.

Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE

BackgroundCancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).ResultsThe stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot.ConclusionThese findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis.

Clonal Selection in Malignant Transformation of Human Fibroblasts Transduced with Defined Cellular Oncogenes

Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations.

Down-Regulation of Overexpressed Sp1 Protein in Human Fibrosarcoma Cell Lines Inhibits Tumor Formation

Sp1 is a transcription factor for many genes, including genes involved in tumorigenesis. We found that human fibroblast cells malignantly transformed in culture by a carcinogen or by stable transfection of an oncogene express Sp1 at 8-fold to 18-fold higher levels than their parental cells. These cell lines form fibrosarcomas in athymic mice with a very short latency, and the cells from the tumors express the same high levels of Sp1. Similar high levels of Sp1 were found in the patient-derived fibrosarcoma cell lines tested, and in the tumors formed in athymic mice by these cell lines. To investigate the role of overexpression of Sp1 in malignant transformation of human fibroblasts, we transfected an Sp1 U1snRNA/Ribozyme into two human cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. The level of expression of Sp1 in these transfected cell lines was reduced to near normal. The cells regained the spindle-shaped morphology and exhibited increased apoptosis and decreased expression of several genes linked to cancer, i.e., epithelial growth factor receptor, urokinase plasminogen activator, urokinase plasminogen activator receptor, and vascular endothelial growth factor. When injected into athymic mice, these cell lines with near normal levels of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. These data indicate that overexpression of Sp1 plays a causal role in malignant transformation of human fibroblasts and suggest that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy.

Defects in translational regulation mediated by the alpha subunit of eukaryotic initiation factor 2 inhibit antiviral activity and facilitate the malignant transformation of human fibroblasts.

Suppression of protein synthesis through phosphorylation of the translation initiation factor alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is known to occur in response to many forms of cellular stress. To further study this, we have developed novel cell lines that inducibly express FLAG-tagged versions of either the phosphomimetic eIF2alpha variant, eIF2alpha-S51D, or the phosphorylation-insensitive eIF2alpha-S51A. These variants showed authentic subcellular localization, were incorporated into endogenous ternary complexes, and were able to modulate overall rates of protein synthesis as well as influence cell division. However, phosphorylation of eIF2alpha failed to induce cell death or sensitize cells to killing by proapoptotic stimuli, though it was able to inhibit viral replication, confirming the role of eIF2alpha in host defense. Further, although the eIF2alpha-S51A variant has been shown to transform NIH 3T3 cells, it was unable to transform the murine fibroblast 3T3 L1 cell line. To therefore clarify this issue, we explored the role of eIF2alpha in growth control and demonstrated that the eIF2alpha-S51A variant is capable of collaborating with hTERT and the simian virus 40 large T antigen in the transformation of primary human kidney cells. Thus, dysregulation of translation initiation is indeed sufficient to cooperate with defined oncogenic elements and participate in the tumorigenesis of human tissue.

Malignant transformation of human fibroblasts by ionizing radiation.

As one step in developing an assay for quantifying the induction of malignant transformation of human cells by ionizing radiation, we exposed cells from a non-tumorigenic, infinite life span, near-diploid fibroblast strain MSU-1.1 to 4.35 Gy 60Co radiation and assayed them for focus formation. The mean frequency of foci in the irradiated population was 6 x 10(-7) cells assayed. No foci were found in the control cells. Of four focus-derived cell strains studied in detail, two produced malignant tumours within 3-7 weeks. The other two did not produce tumours during the 12-month period of study. The tumours from one strain were classified as sarcomas composed exclusively of spindle-shaped cells. Tumours from the other strain were sarcomas consisting of a mixed population of round and spindle cells. Immunoprecipitation analysis of the status of the p53 gene in the focus-derived strains, using a mutant-specific anti-body (Pab240) and an antibody that recognizes both mutant and wild-type p53 protein (Pab421), showed that the tumorigenic strains were completely devoid of p53 protein. One non-tumorigenic strain expressed wild-type p53 protein, and the other expressed a lower molecular weight form of the protein. Karyotypic analysis showed that the tumour-derived cells from one tumorigenic strain had lost one copy of chromosome 6, 14, 16 and 17. The tumour-derived cells from the second strain had lost one copy of chromosome 7, 13, 14 and 17 and part of chromosome 6, as well as part of the other copy of chromosome 7 and 17. These results suggest that the common loss of one copy of chromosome 14, 17 and part of 6 plays a causal role in the malignant transformation of these cells. Furthermore, the results indicate that it will be possible to develop a system that uses near-diploid human fibroblasts to quantify radiation-induced malignant transformation.

Malignant transformation of human fibroblasts previously immortalized with 60Co gamma rays.

Normal human fibroblasts obtained from a whole embryo were malignantly transformed with only 60Co gamma rays, but after extensive passage. The fibroblasts were exposed intermittently to 60Co gamma rays, 13 times, 2,800 rads in total, from the primary culture to the 40th passage level. At the 50th passage level the cells became morphologically changed, showing chromosomal abnormalities, and serial passages showed them to be immortalized. They were not tumorigenic on transplantation into nude mice, but became malignant after extensive passage; i.e., at the 547th passage level and 2,800 days after initiation of the culture. Our results indicate that several different genotypic changes are necessary for malignant transformation of human cells over long periods. No mutation at codons 12 and 61 of H-, K- and N-ras was detected in the malignant cells. Thus, this system may be useful to detect other cellular genes that may contribute to the malignant phenotypes of human fibroblasts.

Malignant transformation of human fibroblasts caused by expression of a transfected T24 HRAS oncogene.

We showed previously that diploid human fibroblasts that express a transfected HRAS oncogene from the human bladder carcinoma cell line T24 exhibit several characteristics of transformed cells but do not acquire an infinite life-span and are not tumorigenic. To extend these studies of the T24 HRAS in human cells, we have utilized an infinite life-span, but otherwise phenotypically normal, human fibroblast cell strain, MSU-1.1, developed in this laboratory after transfection of diploid fibroblasts with a viral v-myc oncogene. Transfection of MSU-1.1 cells with the T24 HRAS flanked by two transcriptional enhancer elements (pHO6T1) yielded foci of morphologically transformed cells. No such transformation occurred if the plasmid containing T24 HRAS had only one enhancer or none at all or if the normal human HRAS gene was transfected in the pHO6 vector (pHO6N1). Cell strains derived from such foci expressed high levels of T24 HRAS product p21, formed colonies in soft agar at high frequency, proliferated rapidly in serum-free medium that does not support growth of the parental cell line, and formed progressively growing, invasive fibrosarcomas. These foci-derived T24 HRAS-transformed cell strains, as well as cells from the tumors derived from them, had the same near-diploid karyotype as that of the parental MSU-1.1 cells. Transfection of pHO6T1 into two other infinite life-span human fibroblast cell lines, cells that had not been transfected with v-myc, also resulted in malignant transformation, suggesting that the infinite life-span phenotype of MSU-1.1 cells, and not necessarily expression of the v-myc oncogene, was the factor that complemented T24 HRAS expression to cause malignant transformation.

Towards an understanding of the malignant transformation of diploid human fibroblasts

This paper reviews the mahor reports of the spontaneous or carcinogen-induced transformation of human fibroblasts to the malignant state, to infinite lifespan, or to anchorage independence. In some cases, the transformed cells and the parent cell with which the work began were made available to us to be tested to determine wether the cell shared common isozymes, HLA antigens, restriction-fragment length polymorphisms, marker chromosomes, etc., as one would expect. When we examined the normal fibroblastic cell line KD for these markers, and the transformed HuT cell lines developed from it by Kakunaga (Proc. Natl. Acad. Sci. (U.S.A.), 75, 1334, 1978) for these markers, we found marked differences, indicating that KD cells and HuT cells are derived from different individuals. When we applied these techniques to the 3 human fibroblast cell lines transformed by Namba to acquire infinite lifespan in culture (Gann, 27, 221, 1981), it became clear that KSMT-6 was derived from the parent cell, KMS-6, but that both cell lines CT-1 and SUSM-1 were derived from the same parental cell line, AD387. Similar studies with other sets of cell lines are also reported. In the light of these studies, it appears that there is no example of the malignant transformation of human fibroblasts by carcinogen treatment. However, neoplastic transformation and transformation to infinite lifespan by carcinogen treatment have been achieved by a number of workers. We speculate as to how malignant transformation might be obtained by carcinogen treatment.

Malignant transformation of human fetal lung fibroblasts induced by nitrosamine compounds in vitro.

This report, for the first time, demonstrates that diethyl-nitrosamine (NDEA) and a new nitrosamine compound-N-1-methylacetonyl-N-3-methylbutyl-nitrosamine (MAMBNA), which are suspicious carcinogens of esophageal cancer (EC) in Linxian, Henan Province, are able to cause the malignant transformation (altered morphology, extended life span, anchorage independent growth, invasiveness, tumorigenicity, etc.) of normal diploid fibroblasts derived from human fetal lung. Besides, the preselection for clonogenic cells, which seem to be more readily to be transformed, in soft agar before carcinogen treatment, could be considered as one of the useful ways in establishment of a model system of malignant transformation of human fibroblasts in vitro.

Changes in gene expression accompanying chemically-induced malignant transformation of human fibroblasts

The modulation of gene expression accompanying neoplastic transformation has been assessed by computerized microdensitometry or autoradiographic patterns of [35S]methionine labeled polypeptides separated by two-dimensional polyacrylamide gel electrophoresis. Nearly 1000 polypeptide species of parent diploid human fibroblasts (KD strain) and clonally-derived malignant fibroblasts (HUT-14 strain) were compared. HUT-14 fibroblasts express a mutation in one of the two functional beta-actin genes and possess properties that distinguish them as neoplastic cells. Of the 700 more abundant polypeptides measured, 13 were lost and 14 were gained following this neoplastic transformation. It is estimated that less than or equal to 2% of the genes expressing abundant polypeptides were either activated or shut off, but at least 32% were modulated quantitatively as a consequence of this neoplastic transformation. Classes of "highly variable" and "marginally variable" polypeptides were assigned. Among the "highly variable" polypeptides, two related species barely detectable in KD parental cells were synthesized at a 25-31-fold higher rate in the transformed cells, and the cell-associated and extracellular matrix forms of fibronectin were each diminished by greater than 90%. Principles emerging from this study may form a basis for interpretation of the role of individual genes in the expression of neoplastic characteristics of HUT-14 cells.