Genetic and biochemical studies on the suppression of and a recovery from the tumorous state in higher plants

Summary Four neoplastic diseases of plants: crown gall, which is caused by Ti plasmid DNA; Black's wound tumor disease by an RNA virus; the Kostoff genetic tumors by chromosomal imbalance; and habituation, which results from a spontaneous activation of select biosynthetic systems, have been analyzed and compared. It has been found that both the development of a capacity for autonomous growth and the nature of the heritable cellular change that underlies tumorigenesis are similar in the four instances. All develop a capacity for autonomous growth as a result of the persistent activation of select biosynthetic systems, the products of which are concerned with cell growth and division. That the persistent activation of these biosynthetic systems does not involve heritable changes of an irreversible type is indicated by the finding that a reversal of the neoplastic state occurred in three of the test systems. Since the tumor cells in these instances were found to remain totipotent the results suggest that whether the normal or tumor phenotype is expressed is determined by how the genetic information is regulated in a cell. Regulation appears to be accomplished in part through positive feedback control mechanisms. Foreign genetic information could act either in a regulatory manner to persistently activate normal biosynthetic systems or it could code for one or more essential but normally limiting substance(s) and thus replace a substance(s) that in the case of the Kostoff tumors or habituation is specified by host cell genes, or it could do both. In either case, the foreign genetic information can be regulated in much the same manner as are the host cell genes to give rise to either the normal or tumor phenotype. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. Certain of the investigations described above were supported in part by Grant Number CA-13808, awarded by the National Cancer Institute, U.S. Department of Health, Education, and Welfare, in which the author is the coprincipal investigator.     

Mutations and epimutations in the origin of cancer

Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

The chemokine system in cancer biology and therapy

Chemokines are a key component of cancer-related inflammation. Chemokines and chemokine receptors are downstream of genetic events that cause neoplastic transformation and are components of chronic inflammatory conditions, which predispose to cancer. Components of the chemokine system affect in a cell autonomous or non-autonomous way multiple pathways of tumor progression, including: leukocyte recruitment and function; cellular senescence; tumor cell proliferation and survival; invasion and metastasis. Available information in preclinical and clinical settings suggests that the chemokine system represents a valuable target for the development of innovative therapeutic strategies.     

Transforming growth factors and the regulation of cell proliferation

The number of different growth regulatory molecules which have been isolated and characterized is continuing to increase. As more information is obtained, it has become apparent that the cooperative actions of many factors with distinct activities is necessary for appropriate proliferative responses. An interplay of both growth stimulatory and growth inhibitory factors is essential for normal growth. Of crucial importance, therefore, is the appropriate regulation of growth factors. Unregulated expression, synthesis, posttranslational processing or activation of either positive or negative growth signals may contribute to neoplastic transformation (Fig. 3). Altered responses to normally positive or negative signals by transformed cells have been demonstrated by several investigators [64, 79, 84]. While altered growth factor responses in transformed cells are well documented, the mechanisms responsible for the loss of growth control are poorly understood and are likely to be both complex and numerous. Continued efforts to dissect and comprehend fully growth factor action on normal cells will be necessary before an understanding of neoplastic transformation can be achieved.     

Transdifferentiation in neoplastic development and its pathological implication

Transdifferentiation is a process in which a cell committed to a particular specialization changes to another quite distinct type. It occurs during embryological development and some pathological processes, and causes the tumor cells to express a phenotype different from that of their normal progenitors. Neoplastic transdifferentiation involves pathogenesis of cancer subtype, transition between neoplastic epithelia and neuroendocrine cell, transition between neoplastic epithelia and mesenchyme, as well as transition between non-neuroectodermal and neuroectodermal cells. We propose that differentiation disturbance of cancer cells should include not only lower-, un-, or de-differentiation, but also transdifferentiation. Tumor cell transdifferentiation results from genetic instabilities. In some type of neoplastic transition, the initiation may be induced by extracellular matrix and growth factors.     

Drosophila genetic variants that change cell size and rate of proliferation affect cell communication and hence patterning

We explore in this paper the role of genetic variants that affect cell size and proliferation in the determination of organ size. We use genetic mosaics of loss or gain of function in six different loci, which promotes smaller or larger than normal cells, associated to either smaller or larger than normal territories. These variants have autonomous effects on patterning and growth in mutant territories. However, there is no correlation between cell size or rate of proliferation on the size of the mutant territory. In addition, these mosaics show non-autonomous effects on surrounding wildtype cells, consisting always in a reduction in number of non-mutant cells. In all mutant conditions the final size (and shape) of the wing is different than normal. The phenotypes of the same variants include higher density of chaetae in the notum. These autonomous and non-autonomous effects suggest that the control of size in the wing is the result of local cell communication defining canonic distances between cells in a positional-values landscape.     

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.     

Habituation: cultural curiosity or developmental determinant?

The induction and reversal of habituation for auxin or cytokinin have been brought about by exposure of callus tissue to low or high concentrations of the growth substance in question. Since the growth rate of the tissues must be measured in the presence or absence of the growth substance being tested, habituation can be demonstrated only in callus. However some developmental processes in the intact plant seem to imply the possible involvement of habituation-like processes in normal development. Evidence for this, and the implications for the action of growth substances in plant development are considered.     

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.     

5-azacytidine-induced tumorous transformation and DNA hypomethylation in Nicotiana tissue cultures

The phenomenon of habituation is considered in plant tissue cultures to be a real process of chemical tumorogenesis; the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca x N. langsdorffii nontumorous hybrid (NNT) during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.     

Interferon-gamma induces altered oncogene expression and terminal differentiation in A431 cells

In tumor cell lines in which oncogene expression is abnormal, modulation of the expression of the oncogene (myc, src, or ras) by interferons (IFNs) has been observed concurrently with cell growth inhibition or phenotypic reversion. Oncogene expression has also been reported to vary during the differentiation of several neoplastic cell lines. Treatment of monolayer cultures of A431, a human epidermoid carcinoma cell line, with IFN-gamma resulted in rapid morphological alterations and cell death not seen with either IFN-alpha or IFN-beta. These changes were accompanied by elevated expression of mRNA's for p21 (the c-ras gene product) and the epidermal growth factor receptor as well as increases in the biosynthetic rate of their respective proteins. These effects likewise appeared to be specific for IFN-gamma. Growth inhibition by IFN-gamma was also observed when A431 cells were grown in a three dimensional in vitro culture system. Immunohistochemical staining of these "tumoroids" with a differentiation specific, anti-keratin antibody indicated that IFN-gamma enhanced expression of this keratin. This observation suggests that the killing by IFN-gamma of A431 cells may result from an acceleration of terminal differentiation.     

Macrophage activation by tuftsin and muramyl-dipeptide

Summary Peritoneal macrophages from tuftsin or MDP-treated mice were tested for their cytostatic activity for tumor cell proliferation. Both substances are able to activate macrophages either after intravenous injection or after incubation in vitro with normal macrophages. But a stimulation as well as an inhibition of tumor cell growth can result from macrophage activation depending on the timing and dose injected. Restoration of the impaired cytostatic capacity of macrophages of mice observed with aging, is obtained by repeated administration of tuftsin. Normal and BCG-stimulated macrophages were examined for their regulatory activity on the proliferation of P815 tumor cells. Low density of macrophages per well determines a stimulation of target cell growth whether the macrophages are normal or activated. When the number of macrophages is increased, under conditions in which normal macrophages are not inhibitory. BCG-stimulated macrophages exert already a strong cytostatic activity. At high macrophage content it appears that normal macrophages can also display an inhibitory activity. Macrophage-tumor cell interactions are highly dependent on the concentration and the state of activation of macrophages.Reprint requests should be addressed to M. Bruley-Rosset     

The multifaceted role of Notch in cancer

The diverse roles that Notch signals play during the development and maintenance of normal tissues are recapitulated in different forms of cancer. Depending on the tumor type, Notch can variously promote or limit tumor growth through either cell autonomous or cell non-autonomous effects on differentiation, cellular metabolism, cell cycle progression, angiogenesis, and possibly self-renewal and immune function. Of particular interest, recent findings indicate that a high fraction of T-cell acute lymphoblastic leukemias and lymphomas have activating mutations in the Notch 1 receptor, and that Notch signaling might have a role in the maintenance of normal and malignant stem cells.