Retrotransposon-like VL30 elements are efficiently induced in anoxic rat fibroblasts

VL30 elements are a multigene family within the class of retroviruses and retrotransposons. We have characterized the response of normal rat fibroblasts to anoxia, in which endogenous VL30 element expression is strongly induced. Optimal induction up to 500-fold occurs under complete anoxia, although a lesser response is seen under atmospheres up to 2% oxygen. Phorbol esters and diacylglycerol, which induce mouse VL30 RNA approximately eightfold, show no effect on the rat VL30 system. The hypoxic conditions optimal for rat VL30 induction represent a mild cellular stress, with no reduction in cell viability during the induction period. Although the precise physiological role of this fibroblast response to temporary anoxia is unknown, it may occur during wound healing. The induction of VL30 by anoxia provides a unique model system wherein a member of the mammalian retrovirus/retrotransposon family is highly responsive to a common physiological signal.     

Anoxia-inducible rat VL30 elements and their relationship to ras-containing sarcoma viruses.

VL30 elements are associated with cancer by their overexpression in rodent malignancies, their induction in a fibroblast response to anoxia which shares features with the malignant phenotype, and their presence recombined into Harvey murine sarcoma virus (HaSV) and Kirsten murine sarcoma virus. These sarcoma viruses contain ras oncogenes flanked on both sides by retrotransposon VL30 element sequences, in turn flanked by mouse leukemia virus sequences. Three very basic questions have existed about the VL30 element sequences found in sarcoma viruses: (i) how did they become recombined, (ii) what are their exact boundaries, and (iii) why are they there? To help decipher the nature of VL30 elements in sarcoma viruses, we examined VL30 clones isolated from an anoxic fibroblast cDNA library and independently by polymerase chain reaction cloning from rat cell DNA. Sequence comparisons with HaSV revealed that HaSV was formed by the substitution of 0.7 kb of VL30 sequences by 0.9 kb of c-Ha-ras sequences, with this event possibly facilitated by the presence of an identical Alu-like repeat found upstream of the 5' recombination point in both the VL30 element and c-Ha-ras. Recombination occurred 42 bases beyond the Alu-like sequences in VL30 and 1596 bases beyond them in c-Ha-ras, at position 926 of HaSV. The 3' ras-VL30 recombination event in HaSV occurred within a seven-base region of shared sequence identity, between HaSV bases 1825 and 1825 and 1831. Recombination between Moloney leukemia virus (MoLV) and VL30 appears to have occurred at a point corresponding to base 218 or 219 of MoLV and was near a TAR-like VL30 sequence; such recombination at the 3' end was between positions 7445 and 7456 of MoLV (HaSV positions 4694 to 4703). Kirsten murine sarcoma virus was found to be closely analogous to HaSV, and limited similar features were also seen with Rasheed sarcoma virus.     

The Wellcome Foundation lecture, 1986. The molecular regulators of normal and leukaemic blood cells

The development of a cell-culture system for the cloning and clonal differentiation of different types of blood cell has made it possible to identify: (i), the proteins that regulate growth and differentiation of different cell lineages in normal and leukaemic blood cells; (ii), the molecular basis of normal and abnormal control of cell development in blood-forming tissue; and (iii), how to suppress malignancy in leukaemic cells. By using myeloid blood cells as a model system, it has been shown that normal blood cells require different proteins to induce cell viability and multiplication (growth-inducers) and differentiation (differentiation-inducers), that there is a hierarchy of growth-inducers which act at various stages of cell development, and that a growth-inducer can switch on production of a differentiation-inducer. Gene cloning has established a multigene family for these proteins. Identification of these proteins and their interaction has shown how growth and differentiation are regulated in normal development and demonstrated the mechanisms that uncouple growth and differentiation so as to produce malignant cells. Normal cells require an external source of growth-inducing protein for cell viability and multiplication. Cells can become leukaemic by genetically changing this normal requirement for growth without blocking response to normal differentiation-inducers. The mature cells induced by adding these normal protein-inducers are then no longer malignant. Other genetic changes which inhibit differentiation by the normal blood-cell regulatory proteins can occur in the evolution of leukaemia. But even these leukaemic cells may still be induced to differentiate by other compounds that can induce differentiation by alternative pathways. The differentiation of leukaemic to mature cells, which stops the cells from multiplying, results in the suppression of malignancy by bypassing genetic changes that produce the malignant phenotype. The activity of blood-cell growth- and differentiation-inducing proteins has been shown in culture and in the body. They can, therefore, be clinically useful to correct defects in the development of normal and leukaemic blood cells.     

Hypoxia-generated superoxide induces the development of the adhesion phenotype

Adhesion fibroblasts exhibit higher TGF-beta1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-beta1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-beta1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-beta1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-beta1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-beta1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-beta1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.     

Heterotypic signaling between dermal fibroblasts and melanoma cells induces phenotypic plasticity and proteome rearrangement in malignant cells

The signaling events triggered by soluble mediators released from both transformed and stromal cells shape the phenotype of tumoral cells and have significant implications in cancer development and progression. In this study we performed an in vitro heterotypic signaling assays by evaluating the proteome diversity of human dermal fibroblasts after stimulation with the conditioned media obtained from malignant melanoma cells. In addition, we also evaluated the changes in the proteome of melanoma cells after stimulation with their own conditioned media as well as with the conditioned medium from melanoma-stimulated fibroblasts. Our results revealed a clear rearrangement in the proteome of stromal and malignant cells upon crosstalk of soluble mediators. The main proteome signature of fibroblasts stimulated with melanoma conditioned medium was related to protein synthesis, which indicates that this process might be an early response of stromal cells. In addition, the conditioned medium derived from ‘primed’ stromal cells (melanoma-stimulated fibroblasts) was more effective in altering the functional phenotype (cell migration) of malignant cells than the conditioned medium from non-stimulated fibroblasts. Collectively, self- and cross-stimulation may play a key role in shaping the tumor microenvironment and enable tumoral cells to succeed in the process of melanoma progression and metastasis. Although the proteome landscape of cells participating in such a heterotypic signaling represents a snapshot of a highly dynamic state, understanding the diversity of proteins and enriched biological pathways resulting from stimulated cell states may allow for targeting specific cell regulatory motifs involved in melanoma progression and metastasis.     

Constitutive connective tissue growth factor expression in scleroderma fibroblasts is dependent on Sp1

Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.     

Cell differentiation and malignancy

An understanding of the mechanism that controls growth and differentiation in normal cells would seem to be an essential requirement to elucidate the origin and reversibility of malignancy. For this approach I have mainly used normal and leukemic blood cells, and in most studies have used myeloid blood cells as a model system. Our development of systems for the in vitro cloning and clonal differentiation of normal blood cells made it possible to study the controls that regulate growth (multiplication) and differentiation of these normal cells and the changes in these controls in leukemia. Experiments with normal blood cell precursors have shown that normal cells require different proteins to induce growth and differentiation. We have also shown that in normal myeloid precursors, growth-inducing protein induces both growth and production of differentiation-inducing protein so this ensures the coupling between growth and differentiation that occurs in normal development. The origin of malignancy involves uncoupling of growth and differentiation. This can be produced by changes from inducible to constitutive expression of specific genes that result in asynchrony to the coordination required for the normal developmental program. Normal myeloid precursors require an external source of growth-inducing protein for growth, and we have identified different types of leukemic cells. Some no longer require and other constitutively produce their own growth-inducing protein. But addition of the normal differentiation-inducing protein to these malignant cells still induces their normal differentiation, and the mature cells are then no longer malignant. Genetic changes that produce blocks in the ability to be induced to differentiate by the normal inducer occur in the evolution of leukemia. But even these cells can be induced to differentiate by other compounds, including low doses of compounds now being used in cancer therapy, that induce the differentiation program by other pathways. This differentiation of leukemic cells has been obtained in vitro and in vivo, and our in vivo results indicate that this may be a useful approach to therapy. In some tumours, such as sarcomas, reversion from a malignant to a non-malignant phenotype can be a result of chromosome changes that suppress malignancy. But in myeloid leukemia, the stopping of growth in mature cells by induction of differentiation bypasses the genetic changes that produce the malignant phenotype. These conclusions can also be applied to other types of normal and malignant cells.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

Preferential expression of a common T cell receptor structure by T cells induced to proliferate in vitro with a murine retrovirus.

We have successfully isolated continuous T cell lines from murine spleen which have been induced to proliferate after in vitro exposure to the murine leukemia virus RadLV. Cell lines isolated from several strains of mice have an "immature" phenotype and are immortalized CD4- CD8- CD3+ cell lines. Cell lines of similar phenotype have now been derived from many individual mice, after spleens have been infected with two different RadLV viruses, a leukemogenic and a nonleukemogenic isolate. Among cell lines induced with RadLV/C6VL, an unusually high proportion of cells was found to bind the 124-40 anticlonotypic antibody specific for the alpha beta TCR expressed by C6VL/1 cells which produces RadLV/C6VL. This was not reflected in cell lines induced with the RadLV/V13 isolate nor in various lymphocyte subsets freshly isolated from normal mice, or induced to proliferate in culture. Cells expressing a common TCR structure would appear to be appropriate targets for in vitro proliferation and transformation induced by RadLV.     

Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

Transforming growth factor-beta(1) (TGF-beta(1)) induces alpha-smooth muscle actin (alpha-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased alpha-SMA expression. Because TGF-beta(1) is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-beta(1) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-beta(1), and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-beta(1) induces not only alpha-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.     

A genetic basis for metastasis.

The progression of a normal cell to one that is malignant is characterized by at least four progressive but potentially separable behavioural patterns identifying the immortal, tumorigenic, invasive and metastatic phenotypes. A multitude of steps appear to be involved in both transformation from normality and progression to malignancy as characterized by the acquisition of metastatic behaviour. Consequently, it seems unlikely that a single gene can directly manifest expression of the metastatic phenotype in normal cells unless it can induced pleiotropic effects. Indeed, a single trait uniquely characterizing the metastatic phenotype has never been identified. The possibility of a single gene suppressing the metastatic phenotype seems much greater. One possible candidate for such a gene is nm23, the expression of which correlates with reduced metastatic potential in several tumours including breast cancer in humans. Although the numbers involved are still small, the correlation of nm23 expression with breast cancer outcome offers potential in using this system as a prognostic aid in clinical diagnosis of this disease. Its possible role as an indicator of metastatic potential in other human tumours remains to be evaluated, although current evidence suggests that it is unlikely to be of use in colon cancer. Further significant progress requires molecular dissection of the mode of action of its product.     

Multifaceted Roles of Interleukin-6 in Adipocyte–Breast Cancer Cell Interaction

Breast cancer is the most common malignancy in women worldwide, with a developmental process spanning decades. The malignant cells recruit a variety of cells including fibroblasts, endothelial cells, immune cells, and adipocytes, creating the tumor microenvironment. The tumor microenvironment has emerged as active participants in breast cancer progression and response to treatment through autocrine and paracrine interaction with the malignant cells. Adipose tissue is abundant in the breast cancer microenvironment; interactions with cancer cells create cancer-associated adipocytes which produce a variety of adipokines that influence breast cancer initiation, metastasis, angiogenesis, and cachexia. Interleukin (IL)-6 has emerged as key compound significantly produced by breast cancer cells and adipocytes, with the potential of inducing proliferation, epithelial-mesenchymal phenotype, stem cell phenotype, angiogenesis, cachexia, and therapeutic resistance in breast cancer cells. Our aim is to present a brief knowledge of IL-6’s role in breast cancer. This review summarizes our current understanding of the breast microenvironment, with emphasis on adipocytes as key players in breast cancer tumorigenesis. The effects of key adipocytes such as leptin, adipokines, TGF-b, and IL-6 are discussed. Finally, we discuss the role of IL-6 in various aspects of cancer progression.