C127 cells resistant to transformation by tyrosine protein kinase oncogenes

C127 is a nontumorigenic mouse cell line widely used in in vitro transformation assays due to its normal morphological appearance and its very low levels of spontaneous transformation. We now report that C127 cells are resistant to transformation by tyrosine protein kinase oncogenes derived from growth factor receptors such as the retroviral v-fms and the human trk transforming genes. In contrast, these cells could be efficiently transformed by members of the ras oncogene family and by serine/threonine kinase oncogenes such as v-mos and v-raf. C127 cells were also found to be resistant to transformation by v-src, the prototype of a large family of tyrosine protein kinase oncogenes whose products are associated with the inner side of the plasma membrane. However, morphologically normal C127 cells expressing pp60v-src acquired a transformed phenotype upon continuous passage in vitro. Somatic cell hybrids (neoR, hygroR) obtained by fusion of G418-resistant C127 cells expressing p70trk (neoR) and hygromycin-resistant NIH3T3 cells (hygroR) exhibited transformed properties as determined by their ability to grow in semisolid agar. In contrast, no such growth was observed when these neoR p70trk-containing C127 cells were fused to control hygroR C127 cells. These results indicate that C127 cells may either lack or express insufficient levels of certain critical substrate(s) necessary for the onset of transformation by tyrosine protein kinase oncogenes.     

Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.     

Transforming mutations in protein-tyrosine kinase genes

Oncogenes are altered forms of normal cellular genes known as proto-oncogenes. Several oncogenes encode enzymes that phosphorylate substrate proteins at tyrosine. In most of these cases the oncogene differs from its proto-oncogene by multiple mutations that alter the structure of the encoded protein product. Here we discuss how structural changes might effect the regulation and substrate specificity of the protein kinase product of a protooncogene so that it gains the potential to transform cells.     

Transformation effector and suppressor genes.

Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto-oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes is referred to by a number of different names including; anti-oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.     

Signal transduction events in mammalian cells in response to ionizing radiation

Ionizing radiations elicit a variety of biological effects in mammalian cells. In recent years altered signal transduction has been recognized as a key cellular response to ionizing radiation. Several oncogenes, the products of which are components of signal transduction pathways and which are over-expressed in many tumors, are specifically induced in cells exposed to radiation. It has also become evident that the oncogene ras and the serine/threonine protein kinase oncogenes raf and PKC confer radio-resistance to tumor cells. Modulation of these genes or their activity by natural compounds may offer a strategy to treat cancer by enhancing radiation-induced apoptosis of tumor cells.     

Immortalization of primary cells by DNA tumor viruses

Cellular senescence is characterized by a decline in sensitivity to growth factors resulting in cessation of cellular growth. The expression of cellular or viral oncogenes may result in the establishment of cell lines with unlimited proliferative potential ("immortalization"). A variety of viral and cellular oncogenes have been reported to immortalize cells, suggesting that multiple mechanisms may lead to an escape from senescence. Immortalization has been reported to occur as a result of an interaction of viral proteins with cellular suppressor gene products or may result from the elevated expression of "transforming" oncoproteins (such as the polyomavirus middle-t antigen). Here we speculate that a selection for cells with a further decreased probability of cell cycle withdrawal can occur during the growth of cells expressing viral early genes, resulting in a process of tumor progression. Explaining immortalization in terms of mitogenic stimulation due to the expression of viral oncogenes followed by genetic/epigenetic changes may help to explain why lytic DNA viruses have a biological activity which may not be necessary for their life cycle.     

Oncogene cooperation in cellular transformation

Oncogenic cell transformation induces major changes in the structure and physiology of the cells: modifications of morphology, differentiation block, disorganisation of cytoskeleton and extracellular matrix, alterations in growth control. The identification of oncogenes relies upon transfer into host normal cells of DNA isolated from cancer cells. The recent development of DNA transfer into germinal cells has provided new insights into the genetic control of tumorogenesis in vivo. In most cases, full transformation into leukemic or tumor cell requires the cooperation of several oncogenes. These observations support the hypothesis of cancer as a multistep process. However, many of the cooperative oncogenes have not yet been identified, especially in human cancers. The recent discovery of genes acting as repressors of cell growth in normal cells has brought to light a new class of potential recessive oncogenes that might have a contributory function in cancer development.     

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.     

Oncogene homologues in yeast

Two different yeasts have a number of genes bearing striking structural and functional homologies to mammalian oncogenes. In yeast these genes are involved in the control of proliferation and early steps in the cell cycle. Many have putative protein kinase activity and some have been shown to control the activity of the enzyme adenylate cyclase which synthesizes cyclic AMP. Mutant forms of these yeast genes have oncogenic activity in mammalian cells.     

Cell cycle control in breast cancer cells

In breast cancer, cyclins D1 and E and the cyclin-dependent kinase inhibitors p21 (Waf1/Cip1)and p27 (Kip1) are important in cell-cycle control and as potential oncogenes or tumor suppressor genes. They are regulated in breast cancer cells following mitogenic stimuli including activation of receptor tyrosine kinases and steroid hormone receptors, and their deregulation frequently impacts on breast cancer outcome, including response to therapy. The cyclin-dependent kinase inhibitor p16 (INK4A) also has a critical role in transformation of mammary epithelial cells. In addition to their roles in cell cycle control, some of these molecules, particularly cyclin D1, have actions that are not mediated through regulation of cyclin-dependent kinase activity but may be important for loss of proliferative control during mammary oncogenesis.     

Malignant cell transformation and oncogenes

In genome of all transforming retroviruses special genes (oncogenes) have been identified which play a key role in malignant conversion of the cells, infected with these viruses. The homologues of these genes (protooncogenes) are persist in all normal cells. During transformation protooncogenes can be activated as a result of one of following processes: insertion of promotor-like elements, mutations, translocations, amplifications or rearrangements. Using transfection technique the transforming genes were isolated from different human tumors. The activation of one of the cellular oncogenes may switch on the other genes and malignant cell transformation may be characterized as a multifactor and multistage process.     

Differential sensitivity to pertussis toxin of 3T3 cells transformed with different oncogenes

Pertussis toxin (PT), which blocks the activity of several G-proteins, has been found to exert a marked inhibitory effect on the DNA synthesis induced in 3T3 cells by serum or growth factors. 3T3 cells transformed with human c-ras oncogenes (Ha-ras, Ki-ras, N-ras) or with src, an oncogene coding for a protein kinase, have lost sensitivity to growth control by PT, even though substrates for PT can still be ADP-ribosylated in vivo. In contrast, 3T3 cells transformed with the SV40 virus behave like normal untransformed cells with respect to the ability of PT to decrease their growth rate. Oncogenes can thus likely be classified either as 'responders' or 'non-responders' to PT.     

人膀胱癌中某些癌基因的研究

本实验应用Northern和斑点印渍杂交技术探测了人膀胱癌细胞株中c-myc、c-fos、erbB等癌基因的表达,以及TPA对这些癌基因表达的调控,发现BIU-87细胞有这些癌基因的表达,并能被TPA所增强,同时也发现人膀胱癌组织有c-myc、c-fos、erbB、N-ras基因的高表达。提示蛋白激酶C的激活可以诱导某些癌基因的表达。多种癌基因的表达异常可能在膀胱癌中起重要作用。     

The gap junction as a “Biological Rosetta Stone”: implications of evolution, stem cells to homeostatic regulation of health and disease in the Barker hypothesis

The discovery of the gap junction structure, its functions and the family of the “connexin” genes, has been basically ignored by the major biological disciplines. These connexin genes code for proteins that organize to form membrane-associated hemi-channels, “connexons”, co-join with the connexons of neighboring cells to form gap junctions. Gap junctions appeared in the early evolution of the metazoan. Their fundamental functions, (e.g., to synchronize electrotonic and metabolic functions of societies of cells, and to regulate cell proliferation, cell differentiation, and apoptosis), were accomplished via integrating the extra-cellular triggering of intra-cellular signaling, and therefore, regulating gene expression. These functions have been documented by genetic mutations of the connexin genes and by chemical modulation of gap junctions. Via genetic alteration of connexins in knock-out and transgenic mice, as well as inherited connexin mutations in various human syndromes, the gap junction has been shown to be directly linked to many normal cell functions and multiple diseases, such as birth defects, reproductive, neurological disorders, immune dysfunction and cancer. Specifically, the modulation of gap junctional intercellular communication (GJIC), either by increasing or decreasing its functions by non-mutagenic chemicals or by oncogenes or tumor suppressor genes in normal or “initiated” stem cells and their progenitor cells, can have a major impact on tumor promotion or cancer chemoprevention and chemotherapy. The overview of the roles of the gap junction in the evolution of the metazoan and its potential in understanding a “systems” view of human health and aging and the diseases of aging will be attempted.     

Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras.

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.